MT-SP1 serine protease

ABSTRACT

This invention provides a novel membrane-type serine protease (designated MT-SP1) elevated expression of which is associated with cancer. In one embodiment, this invention provides a method obtaining a prognosis or of detecting or staging a cancer in an organism. The method involves providing a biological sample from the organism and detecting the level of a membrane type serine protease 1 (MT-SP1) in the sample, where an elevated level of the membrane-type serine protease, as compared to the level of the protease in a biological sample from a normal healthy organism indicates the presence or stage of the cancer.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This work was supported, in part, by National Institutes of HealthGrants Numbers CA72006 and CA71097. The Government of the United Statesof America may have some rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[Not Applicable]

FIELD OF THE INVENTION

This invention relates to the field of serine proteases and associatedbiology. In particular, this invention relates to the discovery of a newmembrane-type serine protease believed to be associated with theetiology of cancer and associated pathologies.

BACKGROUND OF THE INVENTION

The serine proteases (SP) are a large family of proteolytic enzymes thatinclude the digestive enzymes, trypsin and chymotrypsin, components ofthe complement cascade and of the blood-clotting cascade, and enzymesthat control the degradation and turnover of macromolecules of theextracellular matrix. Serine proteases are so named because of thepresence of a serine residue in the active catalytic site for proteincleavage. Serine proteases have a wide range of substrate specificitiesand can be subdivided into subfamilies on the basis of thesespecificities. The main sub-families are trypases (cleavage afterarginine or lysine), aspases (cleavage after aspartate), chymases(cleavage after phenylalanine or leucine), metases (cleavage aftermethionine), and serases (cleavage after serine).

Most proteases are secretory proteins which contain N-terminal signalpeptides that serve to export the immature protein across theendoplasmic reticulum and are then cleaved (von Heijne (1986) Nuc. Acid.Res. 14: 5683-5690). Differences in these signal sequences provide onemeans of distinguishing individual serine proteases. Some serineproteases, particularly the digestive enzymes, exist as inactiveprecursors or preproenzymes, and contain a leader or activation peptidesequence 3′ of the signal peptide. Typically, this activation peptidemay be 2-12 amino acids in length, and it extends from the cleavage siteof the signal peptide to the N-terminal IIGG (SEQ ID NO:76) sequence ofthe active, mature protein. Cleavage of this sequence activates theenzyme. This sequence varies in different serine proteases according tothe biochemical pathway and/or its substrate (Zunino et al. (1988)Biochimica et. Biophysica Acta 967: 331-340; Sayers, et al. (1992) J.Immunology 148: 292-300). Other features that distinguish various serineproteases are the presence or absence of N-linked glycosylation sitesthat provide membrane anchors, the number and distribution of cysteineresidues that determine the secondary structure of the serine proteaseand the sequence of a substrate binding sites such as S′. The S′substrate binding region is defined by residues extending fromapproximately +17 to +29 relative to the N-terminal I (+1). Differencesin this region of the molecule are believed to determine serine proteasesubstrate specificities (Zunino et al, supra).

Numerous disease states are caused by and can be characterized byalterations in the activity of specific proteases and their inhibitors.For example emphysema, arthritis, thrombosis, cancer metastasis and someforms of hemophilia result from the lack of regulation of serineprotease activities (see, for example, Textbook of Biochemistry withClinical Correlations, John Wiley and Sons, Inc. N.Y. (1993)). In caseof viral infection, the presence of viral proteases have been identifiedin infected cells. Such viral proteases include, for example, HIVprotease associated with AIDS and NS3 protease associated with HepatitisC. These viral proteases play a critical role in the virus life cycle.

A series of serine proteases have been identified in murine cytotoxicT-lymphocytes (CTL) and natural killer (NK) cells. These serineproteases are involved with CTL and NK cells in the destruction ofvirally transformed cells and tumor cells and in organ and tissuetransplant rejection (Zunino et al. (1990) J. Immunol. 144: 2001-2009;Sayers et al. (1994) J. Immunol. 152: 2289-2297). Human homologs of mostof these enzymes have been identified (Trapaniet et al. (1988) Proc.Natl. Acad. Sci. 85: 6924-6928; Caputo et al. (1990) J. Immunol. 145:737-744).

Proteases have also been implicated in cancer metastasis. Increasedsynthesis of the protease urokinase has been correlated with anincreased ability to metastasize in many cancers. Urokinase activatesplasmin from plasminogen which is ubiquitously located in theextracellular space and its activation can cause the degradation of theproteins in the extracellular matrix through which the metastasizingtumor cells invade. Plasmin can also activate the collagenases thuspromoting the degradation of the collagen in the basement membranesurrounding the capillaries and lymph system thereby allowing tumorcells to invade into the target tissues (Dano, et al. (1985) Adv.Cancer. Res., 44: 139).

The discovery of a new serine protease precursor and the polynucleotidesencoding it satisfies a need in the art by providing new prognostic anddiagnostic diagnostic methods and, therapeutic compositions useful inthe treatment or prevention of cancer.

SUMMARY OF THE INVENTION

This invention pertains to the discovery of a new serine proteaseassociated with cancer cells. In particular, nucleic acid cDNAs derivedfrom PC-3 mRNA were sequenced that encoded a novel serine proteasereferred to herein as membrane-type serine protease 1 (MT-SP1). TheMT-SP1 polypeptide encoded by the nucleic acid(s) is localized in tumortissues (e.g. prostatic cancers, gastric cancers, breast cancers, etc.),and in preferred embodiments is identified in blood vessels associatedwith tumors. Inhibition of MT-SP1 inhibits cancer growth in relevantanimal models. Without being bound to a particular theory it is believedthat MT-SP1 is implicated in tumor proliferation and/or growth and/ortumor angiogenesis. MT-SP1 is also demonstrated herein to be a gooddiagnostic, and more preferably, a good prognostic for various cancers.MT-SP1 can be used to detect the presence or absence of a cancer, todetermine the location and/or size and/or morphology of a cancer, and tomake a prediction regarding the severity and/or outcome of a cancer or aparticular therapeutic regimen.

In one embodiment, this invention provides nucleic acids encoding MT-SP1and/or probes suitable for amplification of MT-SP1 nucleic acids (e.g.from a PC-3 mRNA template). These nucleic acids include, but are notlimited to: (a) a nucleic acid comprising a nucleic acid encoding aserine protease domain having the sequence of SEQ ID NO: 2; (b) anucleic acid comprising a nucleic acid encoding a serine protease domainhaving the sequence of amino acids 615 through 855 of SEQ ID NO: 2; (c)a nucleic acid that specifically hybridizes to the nucleic acid of SEQID NO: 1 or a fragment thereof under stringent conditions and is ofsufficient length that said nucleic acid can uniquely indicate thepresence or absence of a nucleic acid encoding a membrane-type serineprotease in a total genomic DNA pool, a total cDNA pool or a total mRNApool sample from a PC-3 cell; (d) a nucleic acid comprising a sequencethat has the same sequence as a nucleic acid amplified from a PC-3 cDNAtemplate using PCR primers corresponding to nucleotides 37-54 of SEQ IDNO: 1 and 2604-2583 of the complement of SEQ ID NO: 1; (e) a DNAencoding an mRNA that, when reverse transcribed, produces the cDNA ofSEQ ID NO: 1; (f) a DNA encoding an mRNA that, when reverse transcribed,produces the cDNA encoding amino acids 615-855 of SEQ ID NO: 2; (g) apair of primers that, when used in a nucleic acid amplification reactionwith PC-3 cDNA template specifically amplifies a nucleic acid encodingthe polypeptide of SEQ ID NO: 2; (h) a pair of primers that, when usedin a nucleic acid amplification reaction with mRNA template from a PC-3cell specifically amplify a nucleic acid encoding the polypeptide havingthe sequence of amino acids 615 through 855 of SEQ ID NO: 2; and (i) anucleic acid encoding a membrane-type serine protease, wherein saidnucleic acid encodes a consensus sequence shown in FIG. 4 and does notencode TRYB_human, ENTK-Human, HEPS_human, TRY2_Human, and CTRB_human.Preferred nucleic acids encode a polypeptide having the sequence ofamino acids 615 through 855 of SEQ ID NO: 2, while other preferrednucleic acids encode a polypeptide having the sequence of SEQ ID NO: 2.In one embodiment the nucleic acid has the sequence of SEQ ID NO: 1. Thenucleic acid(s) are optionally present in an expression cassette and/ora vector and are optionally labeled with a detectable label. Alsoprovided are host cells comprising such vectors and a process producinga polypeptide comprising expressing from such host cells a polypeptideencoded an MT-SP1 DNA. This invention also includes a process forproducing a cell that expresses an MT-SP1 polypeptide. The processinvolves comprising transforming or transfecting the cell with thevector encoding an MT-SP1 such that the cell expresses the MT-SP1polypeptide.

In another embodiment this invention provides isolated MT-SP1polypeptides (e.g. as encoded by the nucleic acids described above).Preferred polypeptides comprise a protease domain of SEQ ID NO: 2 or thepolypeptide of SEQ ID NO: 2. Preferred polypeptides also include, butare not limited to polypeptides that have serine protease activity andthat are specifically bound by an antibody raised against thepolypeptide of SEQ ID NO: 2 and/or polypeptides having protease activityand having 95% or greater sequence identity to a polypeptide having thesequence of SEQ ID NO: 2; and/or having protease activity and having 95%or greater identity to a polypeptide having the sequence of amino acids615 through 855 of SEQ ID NO: 2.

Also provided are antibodies that specifically bind to the MT-SP1polypeptides of this invention (e.g. a polypeptide encoded by SEQ ID NO:2). The antibodies can be monoclonal, polyclonal, antibody fragments orsingle-chain antibodies.

This invention also provides diagnostic assays for cancer(s). Suchassays involve providing a biological sample from an organism; anddetecting the level of a membrane type serine protease 1 (MT-SP1) in thesample, where an elevated level of the membrane-type serine protease, ascompared to the level of the protease in a biological sample from anormal healthy organism indicates the presence of the cancer. The methodcan involve determining the copy number of MT-SP1 genes in the cells ofthe biological sample (e.g. using FISH or Comparative GenomicHybridization (CGH)). In another embodiment, the method can involvemeasuring the level of MT-SP1 mRNA in the biological sample, wherein anincreased level of MT-SP1 RNA in the sample compared to MT-SP1 RNA in acontrol sample indicates the presence (or significant probability of thepresence) of the cancer. The mRNA determination can involve hybridizing(e.g. using a Northern blot, a Southern blot, an array hybridization, anaffinity chromatography, an in situ hybridization, etc.) the mRNA to oneor more probes that specifically hybridize (under stringent conditions)to a nucleic acid encoding the MT-SP1 protein. A probe used in suchmeasurements can optionally include a plurality of probes that form anarray of probes. Preferred detection methods involve quantifying MT-SP1mRNA. In still another embodiment, the level of MT-SP1 mRNA is measuredusing a nucleic acid amplification reaction. In addition, oralternatively, the method can involve determining the level (e.g. via amethod selected from the group consisting of capillary electrophoresis,a Western blot, mass spectroscopy, ELISA, immunochromatography, andimmunohistochemistry) or activity of an MT-SP1 protein in the biologicalsample. Preferred biological samples for these assays include, but arenot limited to excised tissue, whole blood, serum, plasma, buccalscrape, saliva, cerebrospinal fluid, and urine.

In certain embodiments, it is desired to pre-screen test agents for theability to bind to an MT-SP1 nucleic acid and/or protein. Suchpre-screening methods typically involve (a) contacting a nucleic acidencoding an MT-SP1 serine protease or an MT-SP1 serine protease proteinwith a test agent; and (b) detecting specific binding of the test agentto the MT-SP1 protein or nucleic acid. Preferred test agents do notinclude antibodies, and/or nucleic acids. In particularly preferredassay formats the MT-SP1 nucleic acid and/or protein is immobilized on asolid support, while in other preferred assay formats, the test agent isimmobilized (e.g. in a 96 well plate, etc.). Preferred methods ofdetecting binding utilize detectable labels (e.g. fluorescent labels)and a particular preferred detection methods utilizes fluorescentresonance energy transfer (FRET).

MT-SP1 levels are also good prognostic indicators for various cancers asdescribed herein. This invention therefore also provides methods(prognostic assays) for evaluating the severity or outcome of a cancer(e.g. for estimating length of survival of a cancer patient). Themethods preferably involve (a) obtaining a biological sample from acancer patient having at least a preliminary diagnosis of cancer; (b)measuring MT-SP1 in said sample and comparing the sample MT-SP1 level tothe MT-SP1 level in normal healthy humans wherein a sample MT-SP1 levelin excess of MT-SP1 levels in normal healthy humans indicates a reducedsurvival expectancy compared to patients with normal MT-SP1 level.Particular embodiments include a preliminary diagnosis of prostatecancer, a cancer of the digestive tract, a breast cancer, and/or aurogential cancer. Preferred biological samples, include, but are notlimited to a primary tumor or a tissue affected by the cancer (e.g. atumor biopsy) and/or samples selected from the group consisting of wholeblood, plasma, serum, synovial fluid, cerebrospinal fluid, bronchiallavage, ascites fluid, bone marrow aspirate, pleural effusion, urine, ortumor tissue. As indicated above, MT-SP1 can be evaluated by copy numberof MT-SP1 genomic DNA, MT-SP1 mRNA levels, levels of nucleic acid(s)derived from MT-SP1 mRNA (e.g. cDNAs, RT-PCR products, etc.), MT-SP1protein levels and/or MT-SP1 activity levels. In a preferred embodimentsthe level of MT-SP1 is measured by immunohistochemical staining of cellscomprising the biological sample (e.g. tumor tissue cells) and/or via animmunoassay (e.g., ELISA using an anti-MT-SP1 antibody as describedabove).

Also provided are methods of treating a cancer in a patient. The methodsinvolve performing one or more of the prognostic assays described hereinon a cancer patient having at least a preliminary diagnosis of a cancer;and (c) selecting a patient identified with an MT-SP1 level excess ofMT-SP1 levels in normal healthy humans and providing an adjuvant cancertherapy (e.g. a therapy selected from the group consisting ofchemotherapy, radiation therapy, reoperation, antihormone therapy, andimmunotherapy).

This invention also affords methods of screening for recurrence of acancer after removal of a primary tumor. These methods involveperforming one or more of the assays described herein on a biologicalsample from a cancer patient following removal of a primary tumor. Theassay can be repeated at a multiplicity of instances after removal ofthe primary tumor.

Similarly the assays of this invention provide methods of monitoring theeffectiveness of cancer treatment in patients. This involves obtaining afirst biological sample from said patient prior to or following one ormore treatments of a cancer; obtaining a second biological sample fromsaid cancer patient during or after said one or more treatments;evaluating the samples for MT-SP1 level as described herein where alower level of MT-SP1 in the second sample as compared to the MT-SP1level in the first sample indicates efficacy of the treatment(s).Typically the treatments involve chemotherapy, radiation therapy,immunotherapy, anti hormone therapy, or surgery.

It was also a discovery of this invention that MT-SP1 provides a goodtarget for specifically delivering an effector (e.g. a liposome, acytotoxin, a detectable label) to a cell (e.g. a tumor cell) expressingMT-SP1. The methods involve providing a chimeric moiety comprising aneffector (e.g. an effector molecule) attached to an anti-MT-SP1antibody; and contacting the cell with said chimeric moiety whereby thechimeric moiety binds (e.g. specifically binds) to the “target” cell. Inpreferred embodiments, the cell (e.g. tumor cell) internalizes at leasta portion (e.g. a detectable or measurable amount) of the molecule.Preferred cells include cancer cells, e.g., cells from a prostatecancer, a cancer of the digestive tract, a breast cancer, and aurogential cancer. Preferred effectors include a cytotoxin, a detectablelabel, a radionuclide, a drug, a liposome, a ligand, and an antibody. Inparticularly preferred embodiments, the chimeric moiety is a fusionprotein. Preferred fusion proteins include effectors selected from thegroup consisting of ricin, abrin, Diphtheria toxin, and Pseudomonasexotoxin. In other preferred embodiments, the effector is cytotoxicand/or a liposome comprising an anti-cancer drug (e.g. doxirubicin,vinblastine, vincristine, taxol, doxirubicin, and genistein).

This invention also provides chimeric moieties (e.g. chimeric molecules)comprising an effector attached to an anti-MT-SP1 antibody. Preferredeffectors include cytotoxin, a detectable label, a radionuclide, a drug,a liposome, a ligand, and an antibody. Preferred chimeric moieties arefusion proteins and particularly preferred fusion proteins havecytotoxic effectors (e.g., ricin, abrin, Diphtheria toxin, Pseudomonasexotoxin, etc.). Other preferred effectors include a liposome comprisingan anti-cancer drug as described above. The chimeric moieties describedherein can be formulated as pharmaceutical compositions comprising thechimeric moiety and pharmaceutically acceptable excipient.

This invention also provides methods of impairing growth of tumor cellsexpressing an MT-SP1 protein. The methods comprise contacting the tumorcells with a chimeric molecule comprising an anti-MT-SP1 antibody; and acytotoxic effector as described herein.

Definitions

The term “nucleic acid” refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, encompasses known analogs of naturalnucleotides that can function in a similar manner as naturally occurringnucleotides.

A “nucleic acid derived from an mRNA transcript” or “nucleic acidderived from an MT-SP1 gene” refers to a nucleic acid for whosesynthesisthe mRNA transcript or a subsequence thereof or the MT-SP1 gene orsubsequence thereof has ultimately served as a template. Thus, a cDNAreverse transcribed from an mRNA, an RNA transcribed from that cDNA, aDNA amplified from the cDNA, an RNA transcribed from the amplified DNA,etc., are all derived from the mRNA transcript and detection of suchderived products is indicative of the presence and/or abundance of theoriginal transcript in a sample. Thus, suitable samples include, but arenot limited to, mRNA transcripts of the gene or genes, cDNA reversetranscribed from the mRNA, cRNA transcribed from the cDNA, DNA amplifiedfrom the genes, RNA transcribed from amplified DNA, and the like.

The terms “isolated” “purified” or “biologically pure” refer to materialwhich is substantially or essentially free from components whichnormally accompany it as found in its native state.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

An amino acid, identified by name herein “e.g., arginine” or “arginineresidue” as used herein refers to natural, synthetic, or version of theamino acids. Thus, for example, an arginine can also include arginineanalogs that offer the same or similar functionality as natural argininewith respect to their ability of be incorporated into a polypeptide,effect folding of that polypeptide and effect interactions of thatpolypeptide with other polypeptide(s).

The phrase “nucleic acid encoding” or “nucleic acid sequence encoding”refers to a nucleic acid that directs the expression of a specificprotein or peptide. The nucleic acid sequences include both the DNAstrand sequence that is transcribed into RNA and the RNA sequence thatis translated into protein. The nucleic acid sequences include bothfull-length nucleic acid sequences as well as shorter sequences derivedfrom the full-length sequences. It is understood that a particularnucleic acid sequence includes the degenerate codons of the nativesequence or sequences which may be introduced to provide codonpreference in a specific host cell. The nucleic acid includes both thesense and antisense strands as either individual single strands or inthe duplex form.

The term “MT-SP1” protease, as used herein refers to either the membranetype serine protease exemplified (e.g. by SEQ ID NOs: 1 and 2) or to theclass of serine proteases characterized by the presence of a non-cleavedsignal/anchor domain that anchors the serine protease in the cellmembrane (see, e.g., Parks & Lamb (1991) Cell 64: 777-787; Parks & Lamb(193) J. Biol. Chem., 268: 19101-19109). Typically, charged residuesflank the sides of the signal/anchor domain was analyzed. Chargedresidues on the N-terminal side of the signal/anchor are important forproper topology, while addition of charges to the C-terminal side of thesignal/anchor has little effect upon orientation. Removal of any of thepositive charges preceding the signal anchor led to partial inversion ofthe topology, suggesting that each positive charge contributes to thesignal. These results indicate that a type II membrane protein ischaracterized by a protein that lacks a cleavable signal sequence (1)and has positively charged residues on the N-terminal side of a longstretch of hydrophobic amino acids (see, e.g., Walter and Lingappa(1986) Annu. Rev. Cell Biol. 2: 499-516).

The term “mutation”, when used in reference to a polypeptide refers tothe change of one or more amino acid residues in a polypeptide toresidues other than those found in the “native” or “reference(pre-mutation) form of that polypeptide. Mutations include amino acidsubstitutions as well as insertions and/or deletions. A mutation doesnot require that the particular amino acid substitution or deletion bemade to an already formed polypeptide, but contemplates that the“mutated” polypeptide can be synthesized de novo, e.g. through chemicalsynthesis or recombinant means. It will be appreciated that the mutationcan include replacement of a natural amino acid with an “unnatural”amino acid.

The term “prognostic” or “prognostic assay” refers to an assay thatprovides an indication as to the outcome of a disease. A prognosticassay need not indicate the presence or absence of a disease. A negativeprognostic assay might indicate the need for a more aggressivetherapeutic regimen.

A “protease” is a polypeptide that cleaves another polypeptide at aparticular site (amino acid sequence). The protease can also beself-cleaving.

A protease is said to be “specific” for another polypeptide when itcharacteristically cleaves the other “substrate” polypeptide at aparticular amino acid sequence. The specificity can be absolute orpartial (i.e., a preference for a particular amino acid or amino acidsequence).

The term “specifically binds” when used to refer to binding proteinsherein indicates that the binding preference (e.g., affinity for thetarget molecule/sequence is at least 2 fold, more preferably at least 5fold, and most preferably at least 10 or 20 fold over a non-specific(e.g. randomly generated molecule lacking the specifically recognizedamino acid or amino acid sequence) target molecule.

The term “phage”, when used in the context of polypeptide display,includes bacteriophage as well as other “infective viruses”, e.g.viruses capable of infecting a mammalian, or other, cell.

The term “chymotrypsin fold” refers to the anti-parallel beta barrelprotein “fold” characteristic of trypsin, chymotrypsin, elastase, andrelated serine proteases (see, e.g., Branden and Tooze (1991)Introduction to Protein Structure, Garland Publishing, New York;Creighton (1993) Proteins, 2nd edition, W.H. Freeman & Co., New York;Schulz and Schirmer (1979) Principles of Protein Structure,Springer-Verlag, New York; Perutz (1992) Protein Structure—NewApproaches to Disease and Therapy, W.H. Freeman & Co., New York; Fersht(1976) Enzyme Structure and Mechanism, 2nd ed., W.H. Freeman & Co., NewYork).

A “protease substrate” is a polypeptide that is specifically recognizedand cleaved by a protease.

The term “modulate” when used with respect to protease activity refersto an alteration in the rate of reaction (protein hydrolysis) catalyzedby a protease. An increase in protease activity results in an increasein the rate of substrate hydrolysis at a particular proteaseconcentration and a protease modulator that produces such an increase inprotease activity is referred to as an “activator” or “proteaseagonist”. The terms “activator” or “agonist” are thus used synonymously.A decrease in protease activity refers to a decrease in the rate ofsubstrate hydrolysis at a particular protease concentration. Such adecrease may involve total elimination of protease activity. A proteasemodulator that produces a decrease in protease activity is referred toas a “protease inhibitor”. It will be appreciated that generally theincrease or decrease is as compared to the protease absent the proteasemodulator.

The term “antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof whichspecifically bind and recognize an analyte (antigen). The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively. An exemplary immunoglobulin(antibody) structural unit comprises a tetramer. Each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). TheN-terminus of each chain defines a variable region of about 100 to 110or more amino acids primarily responsible for antigen recognition. Theterms variable light chain (V_(L)) and variable heavy chain (V_(H))refer to these light and heavy chains respectively.

Antibodies exist e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially an Fab withpart of the hinge region (see, Fundamental Immunology, Third Edition, W.E. Paul, ed., Raven Press, N.Y. 1993). While various antibody fragmentsare defined in terms of the digestion of an intact antibody, one ofskill will appreciate that such fragments may be synthesized de novoeither chemically or by utilizing recombinant DNA methodology. Thus, theterm antibody, as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies, those synthesized denovo using recombinant DNA methodologies (e.g., single chain Fv), andthose found in display libraries (e.g. phage display libraries).

The phrases “hybridizing specifically to” or “specific hybridization” or“selectively hybridize to”, refer to the binding, duplexing, orhybridizing of a nucleic acid molecule preferentially to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA.

The term “stringent conditions” refers to conditions under which a probewill hybridize preferentially to its target subsequence, and to a lesserextent to, or not at all to, other sequences. “Stringent hybridization”and “stringent hybridization wash conditions” in the context of nucleicacid hybridization experiments such as Southern and northernhybridizations are sequence dependent, and are different under differentenvironmental parameters. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I chapter 2 Overview of principles of hybridization and thestrategy of nucleic acid probe assays, Elsevier, N.Y. Generally, highlystringent hybridization and wash conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Very stringentconditions are selected to be equal to the T_(m) for a particular probe.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formamidewith 1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of highly stringent wash conditions is 0.15 M NaClat 72° C. for about 15 minutes. An example of stringent wash conditionsis a 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.) suprafor a description of SSC buffer). Often, a high stringency wash ispreceded by a low stringency wash to remove background probe signal. Anexample medium stringency wash for a duplex of, e.g., more than 100nucleotides, is 1×SSC at 45° C. for 15 minutes. An example lowstringency wash for a duplex of, e.g., more than 100 nucleotides, is4-6×SSC at 40° C. for 15 minutes. In general, a signal to noise ratio of2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Nucleic acids which do not hybridize to each other understringent conditions are still substantially identical if thepolypeptides which they encode are substantially identical. This occurs,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code.

In one particularly preferred embodiment, stringent conditions arecharacterized by hybridization in 1 M NaCl, 10 mM Tris-HCl, pH 8.0,0.01% Triton X-100, 0.1 mg/ml fragmented herring sperm DNA withhybridization at 45° C. with rotation at 50 RPM followed by washingfirst in 0.9 M NaCl, 0.06 M NaH₂PO₄, 0.006 M EDTA, 0.01% Tween-20 at 45°C. for 1 hr, followed by 0.075 M NaCl, 0.005 M NaH₂PO₄, 0.5 mM EDTA at45° C. for 15 minutes.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides, refers to two or more sequences or subsequencesthat have at least 60%, preferably 80%, most preferably 90-95%nucleotide or amino acid residue identity, when compared and aligned formaximum correspondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. Preferably, thesubstantial identity exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably the sequences aresubstantially identical over at least about 150 residues. In a mostpreferred embodiment, the sequences are substantially identical over theentire length of the coding regions.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wisc.), or by visual inspection (see generallyAusubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle (1987) J. Mol. Evol.35:351-360. The method used is similar to the method described byHiggins & Sharp (1989) CABIOS 5: 151-153. The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al, supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g. Karlin & Altschul (1993) Proc. Natl. Acad. Sci.USA, 90: 5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

The term “biological sample” refers to sample is a sample of biologicaltissue, cells, or fluid that, in a healthy and/or pathological state,contains a nucleic acid or polypeptide that is to be detected accordingto the assays described herein. Such samples include, but are notlimited to, cultured cells, primary cell preparations, sputum, amnioticfluid, blood, tissue or fine needle biopsy samples, urine, peritonealfluid, and pleural fluid, or cells therefrom. Biological samples mayalso include sections of tissues (e.g, frozen sections taken forhistological purposes). Although the sample is typically taken from ahuman patient, the assays can be used to detect MT-SP1 in samples fromany mammal, such as dogs, cats, sheep, cattle, and pigs, etc. The samplemay be pretreated as necessary by dilution in an appropriate buffersolution or concentrated, if desired. Any of a number of standardaqueous buffer solutions, employing one of a variety of buffers, such asphosphate, Tris, or the like, at physiological pH can be used.

The term “test agent” refers to an agent that is to be screened in oneor more of the assays described herein. The agent can be virtually anychemical compound. It can exist as a single isolated compound or can bea member of a chemical (e.g. combinatorial) library. In a particularlypreferred embodiment, the test agent will be a small organic molecule.

The term “effector” molecule refers to one or more molecules comprisinga “chimeric molecule or chimeric moiety” whose “activity” it is desiredto deliver (into, adjacent to or in the proximity of) a target cell orcells. The activity need not be activity on the cell, but can simplyprovide a property (e.g. detectability by x-rays, elevatedradiosensitivity, etc.) not normally present at or in the cell. Theeffector while often a single molecule also encompases multi-molecularentities (e.g. liposomes containing drugs, etc.). It is also recognizedthat one or more anti-MT-SP1 antibodies may be attached to any effectoror, conversely, one or more effectors can be attached to a single MT-SP1antibody.

The term “anti-cancer” drug is used herein to refer to one or acombination of drugs conventionally used to treat cancer. Such drugs arewell known to those of skill in the art and include, but are not limitedto doxirubicin, vinblastine, vincristine, taxol, etc.

The term “small organic molecules” refers to molecules of a sizecomparable to those organic molecules generally used in pharmaceuticals.The term excludes biological macromolecules (e.g., proteins, nucleicacids, etc.). Preferred small organic molecules range in size up toabout 5000 Da, more preferably up to 2000 Da, and most preferably up toabout 1000 Da.

The term “conservative substitution” is used in reference to proteins orpeptides to reflect amino acid substitutions that do not substantiallyalter the activity (specificity or binding affinity) of the molecule.Typically conservative amino acid substitutions involve substitution oneamino acid for another amino acid with similar chemical properties (e.g.charge or hydrophobicity). The following six groups each contain aminoacids that are typical conservative substitutions for one another: 1)Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of the cDNA encoding human MT-SP1(SEQ ID NO: 1) and predicted protein sequence (SEQ ID NO: 2). Numberingindicates nucleotide or amino acid residue. Amino acids are shown insingle-letter code. The termination codon is shown by an asterisk (*).The underlined stop codon at nucleotide 10 is in frame with theinitiating methionine. The Kozak consensus sequence (Kozak (1991) J.Cell Biol. 115: 887-903) at the start codon is underlined at nucleotide32. The predicted N-glycosylation sites at amino acids 109, 302, 485,and 772 are underlined. A possible polyadenylation sequence (Nevins(1983) Ann. Rev. Biochem. 52: 441-466) at nucleotide 3120 is alsounderlined. The catalytic triad in the serine protease domain ishighlighted: His656, Asp711 and Ser805.

FIG. 2, lane 1 shows the PCR products obtained using degenerate primersdesigned from the consensus sequences flanking the catalytic histidine(5′ His-primer) and the catalytic serine (3′ Ser-primer). The productsremaining between 400 and 550 bp after digestion with BamHI werereamplified using the same degenerate primers. The products from thissecond PCR are shown in lane 2.

FIG. 3 shows the domain structure of human MT-SP1 compared with thedomain structure of hepsin (Leytus et al. (1988) Biochemistry 27:1067-1074) and enteropeptidase (Kitamoto et al. (1994) Proc. Natl. Acad.Sci. USA 91: 7588-7592). SA represents a possible signal anchor, CUBrepresents a repeat first identified in complement components C1r andC1s, the urchin embryonic growth factor and bone morphogenetic protein 1(Bork and Beckmann (1993) J. Mol. Biol. 231: 539-545), L representslow-density lipoprotein receptor repeat (Krieger and Herz (1994) Annu.Rev. Biochem. 63: 601-637), SP represents a chymotrypsin family serineprotease domain (Perona and Craik (1997) J. Biol. Chem. 272:29987-29990), MAM represents a domain homologous to members of a familydefined by meprin, protein A5, and the protein tyrosine phosphatase μ(Beckmann and Bork (1993) Trends Biochem. Sci. 18: 40-41), and MSCRrepresents a macrophage scavenger receptor cysteine-rich motif (Kriegerand Herz (1994) Annu. Rev. Biochem. 63: 601-637). The predicteddisulfide linkages are shown labeled as C—C.

FIGS. 4A, 4B, and 4C show multiple sequence alignments of MT-SP1structural motifs. L represent loops, □ represent beta sheets, □represent alpha helices, and S—S represent disulfides. FIG. 4A showsmultiple sequence alignment of the serine protease domain of MT-SP1 withhuman trypsinogen B (Emi et al. (1986) Gene 41: 305-310), humanenterokinase (Kitamoto et al. (1995) Biochemistry 34: 4562-4568), humanhepsin (Leytus et al. (1988) Biochemistry 27: 1067-1074), human tryptase2 (Vanderslice et al. (1990) Proc. Natl. Acad. Sci. USA 87: 3811-3815),and human chymotrypsinogen B (Tomita et al. (1989) Biochem. Biophys.Res. Commun. 158: 569-575), using standard chymotrypsin numbering.Conserved catalytic and structural residues described in the text areunderlined. FIG. 4B shows alignment of MT-SP1 LDLR with domains of theLDL receptor (Sudhof et al. (1985) Science 228: 815-822). FIG. 4C showsalignment of the CUB domains of MT-SP1 with those found in humanenterokinase (Kitamoto et al. (1995) Biochemistry 34: 4562-4568), humanbone morphogenetic protein 1 (Wozney et al. (1988) Science 242:1528-1534), and complement component C1R (Leytus et al. (1986)Biochemistry 25: 4855-4863).

FIGS. 5A and 5B show the tissue distribution of MT-SP1 mRNA levels.Northern blots of human poly(A)+ RNA from assorted human tissues washybridized with radiolabeled cDNA probes as described under Materialsand Methods. The upper panel shows hybridization using a MT-SP1 1.3 kBcDNA fragment derived from EST clone w39209 and exposed overnight. Thelower panel shows the same blot after being stripped and rehybridizedwith a loading standard (FIG. 5A) β-actin or (FIG. 5B) humanglyceraldehyde phosphate dehydrogenase (GAPDH) cDNA probe exposed fortwo hours. The mobility of RNA size standards are indicated at the left.

FIGS. 6A and 6B show activation and purification of His-tagged MT-SP1protease domain. A representative experiment is shown in (FIG. 6A) and(FIG. 6B). FIG. 6A: Activation at 4° C. is monitored using SDS-PAGE. Theupper band represents inactivated protease domain, and the lower bandrepresents active protease (also verified by N-terminal sequencing).FIG. 6B: The activation of the protein was monitored usinghexahydrotyrosyl-glycyl-arginyl-paranitroanilide as a syntheticsubstrate for the protease domain. (C) Inactive Ser⁸⁰⁵ Ala proteasedomain is cleaved with 10 nM activated His-tagged MT-SP1 protease domainat 37° C. The specific cleavage of active MT-SP1 protease domain isrequired for proper processing at the activation site. Active proteasedomain is shown in lane 7 (+), and no cleavage of the untreated inactiveprotease domain is observed (lane 8, −).

DETAILED DESCRIPTION

This invention pertains to the discovery of novel membrane-type serineproteases whose inhibition results in inhibition of mouse and ratprostate differentiation and the retardation of growth of human PC-3TRAMP prostatic cancer cells. The prototypical protease of thisinvention is referred to herein as membrane-type serine protease 1(MT-SP1).

The cloning and characterization of the MT-SP1 cDNA showed that itencodes a mosaic protein that contains a transmembrane signal anchor,two CUB domains, four LDLR repeats, and a serine protease domain.Northern blotting showed broad expression of MT-SP1 in a variety ofepithelial tissues with high levels of expression in the humangastrointestinal tract and the prostate. In particular MT-SP1 showedsignificant expression in the endothelium of tumor blood vessels

The data presented herein indicate that expression of the MT-SP1membrane-type serine protease(s) are associated with the presence of, orproclivity to, cancer. In particular, without being bound to a theory,it is believed that the membrane-type serine protease MT-SP1participates in a proteolytic cascade that results in cell growth and ordifferentiation. Another structurally similar membrane-type serineprotease, enteropeptidase (FIG. 3), is involved in a proteolytic cascadeby which activation of trypsinogen leads to activation of downstreamintestinal proteases (Huber and Bode (1978) Acc. Chem. Res. 11:114-122). Enteropeptidase is expressed only in the enterocytes of theproximal small intestine thus precisely restricting activation oftrypsinogen. Thus, in contrast to secreted proteases that may diffusethroughout the organism, the membrane association of MT-SP1 allows theproteolytic activity to be precisely localized, which may is importantfor proper physiological function. Improper localization of the enzymeor levels of downstream substrates could lead to disease.

We have found subcutaneous coinjection of PC-3 cells with wild-typeecotin or ecotin M84R/M85R led to a decrease in the primary tumor sizecompared to animals in whom PC-3 cells and saline were injected. Sincewild-type ecotin is a poor, micromolar inhibitor of uPA, serineproteases other than uPA (e.g., an MT-SP1) are believed to be involvedin this primary tumor proliferation. Both wild-type ecotin and ecotinM84R/M85R are potent, subnanomolar inhibitors of MT-SP1, stronglysuggesting that MT-SP1 plays an important role in progression ofepithelial cancers expressing this protease.

In addition, MT-SP1 is associated with tumors (e.g. is a good tumormarker). In particular, immunohistochemical examination of gastriccancer tissue revealed MT-SP1 expression in cancer cells, endothelialcells and some leukocytes. In these tissues, endothelial cells showedespecially intensive MT-SP1 immunoreactivity indicating that MT-SP1might play an important role in vascular cells particularly inangiogenesis of tumor and tumor-related blood vessels.

In addition, overall survival for groups of gastric carcinoma patientswith highly MT-SP1 expressing endothelium revealed poor prognosiscompared to those with low or no MT-SP1. Higher MT-SP1 expression inendothelium was significantly associated with lower survival rate.

MT-SP1 thus appears to be a good diagnostic, prognostic, or therapeutictarget. In one embodiment, this invention therefore provides methods ofscreening for (e.g. diagnosing) the presence or absence of a cancer bydetecting the level of MT-SP1 expression and/or activity. In aparticularly preferred embodiment this invention provides methods ofscreening for (e.g. diagnosing) the presence of a metastatic cell.

In another embodiment, this invention provides prognostic methods, e.g.,methods of estimating length of survival of a cancer patient, orevaluating the severity of disease or the likilihood of diseaserecurrence in a cancer patient. These methods also involve determiningthe level of MT-SP1 expression and/or activity, where higher expressionlevels indicate greater disease severity, poorer outcome or greaterliklihood of disease recurrence.

MT-SP1 also provides a convenient diagnostic tag for localizing a tumoror cancer cell. This involves providing anti-MT-SP1 antibodies attachedto a detectable tag (e.g. radioactive or radiopaque tage). The labeledMT-SP1 antibody will localize on the surface of tumor cells expressingMT-SP1 and detection of the tag provides an indication fo the presence,locality, and size of the cancer.

Having identified a novel protease involved with the etiology ofcancers, particularly invasive cancers, the MT-SP1 DNA, mRNA, andprotein product provide good targets for the action of putativemodulators. This invention thus, also provides methods of screening formodulators of MT-SP1 expression and/or activity. In addition simplebinding assays can be used to identify agents (e.g. small organicmolecules, antisense molecules, ribozymes, antibodies, etc.) thatinteract specifically with the MT-SP1 nucleic acids and/or proteins.Screening agents for such specific binders identifies putative agentslikely to modulate MT-SP1 expression and/or activity. Collections ofsuch agents provide “biases” molecular libraries that are usefulcandidates for a variety of screening systems.

The localization of MT-SP1 in tumors, and in particular, on vascularendothelial cells associated with tumors provides a convenient methodfor specifically delivering an effector (e.g. an effector molecule) tosuch cells. The method involves attaching one or more molecules thatspecifically bind to MT-SP1 (e.g. an anti-MT-SP1 antibody) to theeffector it is desired to deliver and administering the composition tothe subject (e.g. intraperioneally, intravenously, direct injection intotumor site, etc.). The chimeric antibody-effector composition willlocalize at the tumor site or on cells (e.g. tumor or metastatic cellsexpressing MT-SP1).

This invention also provides MT-SP1 nucleic acids and isolated (e.g.recombinantly expressed) proteins. Such isolated proteins are usefulspecific proteases in their own right. In addition they can be used tohelp “dissect” metabolic pathways and/or can be used as effectiveimmunogens to raise anti-MT-SP1 antibodies.

I. Nucleic Acids Encoding Membrane-Type Serine Proteases.

Using the information provided herein, (e.g. MT-SP1 cDNA sequence,primers, etc.) the nucleic acids (e.g., encoding full length MT-SP1 orMT-SP1 proteolytic domain or other subsequences of the MT-SP1 cDNA,genomic DNA, mRNA, etc). are prepared using standard methods well knownto those of skill in the art. For example, the MT-SP1 nucleic acid(s)may be cloned, or amplified by in vitro methods, such as the polymerasechain reaction (PCR), the ligase chain reaction (LCR), thetranscription-based amplification system (TAS), the self-sustainedsequence replication system (SSR), etc. A wide variety of cloning and invitro amplification methodologies are well-known to persons of skill.Examples of these techniques and instructions sufficient to directpersons of skill through many cloning exercises are found in Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.);Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel); Cashionet al., U.S. Pat. No. 5,017,478; and Carr, European Patent No.0,246,864. Examples of techniques sufficient to direct persons of skillthrough in vitro amplification methods are found in Berger, Sambrook,and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202;PCR Protocols A Guide to Methods and Applications (Inis et al. eds)Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson(Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94;(Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al.(1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J.Clin. Chem., 35: 1826; Landegren et al., (1988) Science, 241: 1077-1080;Van Brunt (1990) Biotechnology, 8: 291-294; Wu and Wallace, (1989) Gene,4: 560; and Barringer et al. (1990) Gene, 89: 117.

The isolation and expression of an MT-SP1 nucleic acid is illustrated inExample 1. In one preferred embodiment, the MT-SP1 cDNA can be isolatedby routine cloning methods. The cDNA sequence provided in SEQ ID NO: 1can be used to provide probes that specifically hybridize to the MT-SP1gene, in a genomic DNA sample, or to the MT-SP1 mRNA, in a total RNAsample (e.g., in a Southern blot). Once the target MT-SP1 nucleic acidis identified (e.g., in a Southern blot), it can be isolated accordingto standard methods known to those of skill in the art (see, e.g.,Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed.,Vols. 1-3, Cold Spring Harbor Laboratory; Berger and Kimmel (1987)Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques,San Diego: Academic Press, Inc.; or Ausubel et al. (1987) CurrentProtocols in Molecular Biology, Greene Publishing andWiley-Interscience, New York). Methods of screening human cDNA librariesfor the MT-SP1 are provided in Example 1.

In another preferred embodiment, the human MT-SP1 cDNA can be isolatedby amplification methods such as polymerase chain reaction (PCR). In apreferred embodiment, the MT-SP1 sequence is amplified from a cDNAsample (e.g., double stranded placental cDNA (Clontech)) using theprimers routinely derived from the sequence illustrated in FIG. 1 (SEQ.ID NO: 1). Preferred primers are primer 1, nucleotides 37-54 of SEQ IDNO: 1) and primer 2, nucleotides 2604-2583 of the complement of SEQ IDNO: 1. Preferred amplification conditions include 30 cycles of 1 minutedenaturing at 94° C., 1 minute annealing at 54° C., 3 minutes ofextension at 72° C., folowed by a final 15 minute extension at 72° C.Preferred template includes full length PCIII cDNA.

Where the MT-SP1 gDNA, cDNA, mRNA or their subsequences are to be usedas nucleic acid probes, it is often desirable to label the nucleic acidswith detectable labels. The labels may be incorporated by any of anumber of means well known to those of skill in the art. However, in onepreferred embodiment, the label is simultaneously incorporated duringthe amplification step in the preparation of the sample nucleic acids.Thus, for example, polymerase chain reaction (PCR) with labeled primersor labeled nucleotides will provide a labeled amplification product. Inanother preferred embodiment, transcription amplification using alabeled nucleotide (e.g. fluorescein-labeled UTP and/or CTP)incorporates a label into the transcribed nucleic acids.

Alternatively, a label may be added directly to an original nucleic acidsample (e.g., mRNA, polyA mRNA, cDNA, etc.) or to the amplificationproduct after the amplification is completed. Means of attaching labelsto nucleic acids are well known to those of skill in the art andinclude, for example nick translation or end-labeling (e.g. with alabeled RNA) by kinasing of the nucleic acid and subsequent attachment(ligation) of a nucleic acid linker joining the sample nucleic acid to alabel (e.g., a fluorophore). Suitable labels are described below.

II. Cloning and Expression of Membrane-Type Serine Proteases.

It is often desirable to provide isolated membrane-type serine proteasesof this invention (e.g., MT-SP1). These polypeptides can be used toraise an immune response and thereby generate antibodies specific to theintact MT-SP1 or to various subsequences or domains thereof. Asexplained below, the MT-SP1 polypeptides and various fragments thereofcan be conveniently produced using synthetic chemical syntheses orrecombinant expression methodologies. In addition to the intactfull-length MT-SP1 polypeptide, in some embodiments; it is oftendesirably to express immunogenically relevant fragments (e.g. fragmentsthat can be used to raise specific anti-MT-SP1 antibodies). In otherpreferred embodiments, the protein is expressed as an inactive form (azymogen or pro-enzyme) that is activated, e.g. via autocleavage, oralternatively, the enzymatic (proteolytic) domain can be expressedalone.

A) De Novo Chemical Synthesis.

The MT-SP1 serine protease precursors, the catalytic domain (activeprotease), or other subsequences of the MT-SP1 polypeptide(s) may besynthesized using standard chemical peptide synthesis techniques. Wherethe desired subsequences are relatively short (e.g., when a particularantigenic determinant is desired) the molecule may be synthesized as asingle contiguous polypeptide. Where larger molecules are desired,subsequences can be synthesized separately (in one or more units) andthen fused by condensation of the amino terminus of one molecule withthe carboxyl terminus of the other molecule thereby forming a peptidebond.

Solid phase synthesis in which the C-terminal amino acid of the sequenceis attached to an insoluble support followed by sequential addition ofthe remaining amino acids in the sequence is the preferred method forthe chemical synthesis of the polypeptides of this invention. Techniquesfor solid phase synthesis are described by Barany and Merrifield,Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA., Merrifield, et al. (1963) J. Am. Chem. Soc., 85: 2149-2156, andStewart et al. (1984) Solid Phase Peptide Synthesis, 2nd ed. PierceChem. Co., Rockford, Ill.

B) Recombinant Expression.

In a preferred embodiment, the MT-SP1 proteins or subsequences thereof(e.g. proteolytic domain), are synthesized using recombinant expressionsystems. Generally this involves creating a DNA sequence that encodesthe desired protein, placing the DNA in an expression cassette under thecontrol of a particular promoter, expressing the protein in a host,isolating the expressed protein and, if required, renaturing theprotein.

DNA encoding the MT-SP1 proteins described herein can be prepared by anysuitable method as described above, including, for example, cloning andrestriction of appropriate sequences or direct chemical synthesis bymethods such as the phosphotriester method of Narang et al. (1979) Meth.Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979)Meth. Enzymol. 68: 109-151; the diethylphosphoramidite method ofBeaucage et al. (1981) Tetra. Lett., 22: 1859-1862; and the solidsupport method of U.S. Pat. No. 4,458,066.

Chemical synthesis produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Alternatively, subsequences may be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments may then be ligated to produce the desired DNA sequence.

In one embodiment, the MT-SP1 nucleic acids of this invention can becloned using DNA amplification methods such as polymerase chain reaction(PCR) (see, e.g., Example 1). Thus, for example, the nucleic acidsequence or subsequence is PCR amplified, using a sense primercontaining one restriction site (e.g., NdeI) and an antisense primercontaining another restriction site (e.g., HindIII). This will produce anucleic acid encoding the desired MT-SP1 sequence or subsequence andhaving terminal restriction sites. This nucleic acid can then be easilyligated into a vector containing a nucleic acid encoding the secondmolecule and having the appropriate corresponding restriction sites.Suitable PCR primers can be determined by one of skill in the art usingthe sequence information provided in SEQ ID NOs: 1 and 2 andrepresentative primers are provided herein. Appropriate restrictionsites can also be added to the nucleic acid encoding the MT-SP1 proteinor protein subsequence by site-directed mutagenesis. The plasmidcontaining the MT-SP1 sequence or subsequence is cleaved with theappropriate restriction endonuclease and then ligated into the vectorencoding the second molecule according to standard methods.

The nucleic acid sequences encoding MT-SP1 proteins or proteinsubsequences may be expressed in a variety of host cells, including E.coli, other bacterial hosts, yeast, and various higher eukaryotic cellssuch as the COS, CHO and HeLa cells lines and myeloma cell lines. Therecombinant protein gene will be operably linked to appropriateexpression control sequences for each host. For E. coli this includes apromoter such as the T7, trp, or lambda promoters, a ribosome bindingsite and preferably a transcription termination signal. For eukaryoticcells, the control sequences will include a promoter and often anenhancer (e.g., an enhancer derived from immunoglobulin genes, SV40,cytomegalovirus, etc.), and a polyadenylation sequence, and may includesplice donor and acceptor sequences.

The plasmids of the invention can be transferred into the chosen hostcell by well-known methods such as calcium chloride transformation forE. coli and calcium phosphate treatment or electroporation for mammaliancells. Cells transformed by the plasmids can be selected by resistanceto antibiotics conferred by genes contained on the plasmids, such as theamp, gpt, neo and hyg genes.

Once expressed, the recombinant MT-SP1 protein(s) can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, (1982) ProteinPurification, Springer-Verlag, N.Y.; Deutscher (1990). Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y.). Substantially pure compositions of at least about 90 to 95%homogeneity are preferred, and 98 to 99% or more homogeneity are mostpreferred. Once purified, partially or to homogeneity as desired, thepolypeptides may then be used (e.g., as immunogens for antibodyproduction). The cloning and expression of a MT-SP1 polypeptides isillustrated in Example 1.

In a preferred embodiment, the MT-SP1 nucleic acid(s) are transformedinto E. coli X-90 to afford high-level expression of recombinantprotease gene products (Evnin et al. (1990) Proc. Natl. Acad. Sci. USA87, 6659-6663). Expression and purification of the recombinant enzymefrom solubilized inclusion bodies is performed according to the methodof Unal et al. (1997) J. Virol. 71, 7030-7038.

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the MT-SP1 protein(s) maypossess a conformation substantially different than the nativeconformations of the constituent polypeptides. In this case, it may benecessary to denature and reduce the polypeptide and then to cause thepolypeptide to re-fold into the preferred conformation. Methods ofreducing and denaturing proteins and inducing re-folding are well knownto those of skill in the art (see, e.g., Debinski et al. (1993) J. Biol.Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4:581-585; and Buchner, et al., (1992) Anal. Biochem., 205: 263-270).Debinski et al., for example, describes the denaturation and reductionof inclusion body proteins in guanidine-DTE. The protein is thenrefolded in a redox buffer containing oxidized glutathione andL-arginine.

One of skill would recognize that modifications can be made to theMT-SP1 proteins without diminishing their biological activity. Somemodifications may be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids (e.g., poly His) placed oneither terminus to create conveniently located restriction sites ortermination codons or purification sequences.

III. Diagnostics/Prognostics—Assays of Membrane-Type Serine ProteaseLevel or Activity.

A) Diagnostic Applications.

MT-SP1 provides an effective marker for the detection/diagnosis of awide variety of cancers. Diagnosis of disease based on measured levelsof MT-SP1 can be made by comparison to levels measured in a disease-freecontrol group or background levels measured in a particular patient. Thediagnosis can be confirmed by correlation of the assay results withother signs of disease known to those skilled in the clinical arts, suchas the diagnostic standards for breast cancer, gastric cancer, prostatecancer, etc.

Because in certain instances serum MT-SP1 may stem from sources otherthan the tissue of interest, in certain embodiments, a sample ispreferably taken from the tissue of interest. However, as describedbelow, in many instances basic differential diagnosis allowsidentification of the pathology resulting in elevated serum MT-SP1.

Particularly for the diagnosis and monitoring of cancers (e.g., tumormetastasis), the preferred source for the assay sample will be blood orblood products (e.g. plasma and/or serum) and/or tissue biopsies. Thoseof ordinary skill in the art will be able to readily determine whichassay sample source is most appropriate for use in diagnosis of aparticular disease for which MT-SP1 is a marker.

The levels of MT-SP1 that are indicative of the development oramelioration of a particular cancer by disease and, to a lesser extent,by patient. Appropriate background MT-SP1 levels in particular tissues,pathologies, and patients or patient populations or control populationscan be determined by routine screening according to standard methodswell known to those of skill in the art.

For purposes of diagnosing the onset, progression, or amelioration ofdisease, variations in the levels of MT-SP1 of interest will be thosewhich differ by a statistically significant level from the normal (i.e.,healthy) population or from the level measured in the same individual ata different time, and which correlate to other clinical signs of diseaseoccurrence and/or prognosis and/or amelioration known to those skilledin the clinical art pertaining to the disease of interest.

Thus, in general, any diagnosis or prognosis indicated by MT-SP1measurements made according to the methods of the invention will beindependently confirmed with reference to clinical manifestations ofdisease known to practitioners of ordinary skill in the clinical arts.

B) Prognostic Applications.

In prognostic applications, MT-SP1 levels are evaluated to estimate therisk of recurrence of a cancer and thereby provide information thatfacilitates the selection of treatment regimen. Without being bound to aparticular theory, it is believed that tumors are heterogeneous (evenwithin a particular tumor type, e.g. colorectal cancer) with respect toelevated expression of MT-SP1. Those tumor types resulting in elevatedlevels of MT-SP1 also show a high likelihood of recurrence, e.g. afterremoval of a primary tumor. Thus, measurement of MT-SP1 levels (before,during [i.e. in blood or tissues removed during surgery], or afterprimary tumor removal) provides a prognostic indication of thelikelihood of tumor recurrence. Where pathologies show elevated MT-SP1levels (e.g. as compared to those in normal healthy subjects) moreaggressive adjunct therapies (e.g. chemotherapy and/or radiotherapy) maybe indicated.

By way of further example, in gastric cancer stages III, IV, patients(n=30) with an MT-SP LI of 40% or higher had a significantly lowersurvival rate than those (n=11) without MT-SP1 expression in vascularcells of cancer tissues. In addition, overall survival for groups ofgastric carcinoma patients with highly MT-SP1 expressing endotheliumrevealed poor prognosis compared to those with low or no MT-SP1. HigherMT-SP1 expression in endothelium was significantly associated with lowersurvival rate indicating that MT-SP1 expression in endothelium aroundcancer cells is an important prognostic factor in gastric cancer.

C) Evaluation of Treatment Efficacy.

The MT-SP1 markers of this invention can also be used to evaluatetreatment efficacy (e.g. amelioration of one or more symptoms of acancer). Where the amelioration of a disease (such as cancer) can berelated to reduction in levels of MT-SP1, MT-SP1 levels in a biologicalassay sample taken from the patient (e.g., blood) can be measured before(for background) and during or after (e.g., at a designated time,periodically or randomly) the course of treatment. Because reductions inMT-SP1 levels may be transient, the assay will preferably be performedat regular intervals, (e.g., every 4 weeks, every 6 months, every year,etc.) closely before and after each treatment. Depending on the courseof treatment, tumor load and other clinical variables, clinicians ofordinary skill in the art will be able to determine an appropriateschedule for performing the assay for diagnostic or disease/treatmentmonitoring purposes.

Such monitoring methods can provide useful information to guide atherapeutic regimen in a variety of contexts as explained below.

1) Checking for Recurrence of a Cancer.

In one embodiment, MT-SP1 is monitored simply to check for the possiblerecurrence of a cancer after the primary tumor has been removed. Thismethod generally involves obtaining a biological sample from a cancerpatient following removal of a primary tumor; and measuring the level ofMT-SP1 in the sample. An elevated MT-SP1 level (e.g. as compared to theMT-SP1 level in normal healthy humans) indicates a possible recurrenceof a cancer. Where patients have elevated MT-SP1 levels at the time ofsurgery, the subsequent MT-SP1 monitoring is most informative after aperiod of time sufficient to permit MT-SP1 levels to return to normal(e.g. about 3-4 weeks after surgery). Of course, monitoring can beperformed earlier to initiate tracking of changes in MT-SP1 levels.Where the patient does not have an elevation in MT-SP1 at the time ofsurgery increased MT-SP1 levels at any time after surgery indicatepossible recurrence of the cancer. Elevated vlevels can be evaluatedrelative to levels in normal healthy people, or relative to MT-SP1baseline levels determined for the particular patient (e.g., prior to,during, or immediately after surgery).

2) Monitoring of Terminal Phase Patients.

In another embodiment, MT-SP1 monitoring can be used to monitor theeffectiveness of cancer treatment in patients with elevated MT-SP1. Suchmonitoring is particularly useful in patients in the terminal phasewhere the cancer has already metastasized so that surgery will notcompletely eliminate the cancer. Such patients will still be treatedwith radiation, chemotherapy, etc, to give them additional months ofsurvival (although in many cases no cure). Periodic measurement ofMT-SP1 provides the clinician with a means of monitoring the progress oftreatment.

3) Checking the Efficacy of Surgical Removal of a Primary Tumor.

In still another embodiment, MT-SP1 monitoring can be used to check forthe effectiveness of surgical removal of a primary tumor, in thoseinstances in which there is an elevation in MT-SP1 prior to surgery.Since our longitudinal study shows that removal of the primary tumorcauses the elevated MT-SP1 levels to fall to normal, measurement ofMT-SP1 in post operative blood (e.g., about 4 weeks after surgery) willreveal those instances in which surgery did not remove all of theprimary tumor, affected lymph nodes, and any other metastasis sites.

D) Relevant Pathologies.

As indicated above, MT-SP1 provides an effective marker for detectionand/or evaluation of prognosis of a wide variety of cancers including,but not limited to, gastric cancer, prostate cancer, cancers of theurinary tract, lung cancer, bronchus cancer, a colorectal cancer (cancerof the colon and/or rectum), breast cancer, pancreas cancer, brain orcentral nervous system cancer, peripheral nervous system cancer,esophageal cancer, cervical cancer, melanoma, uterine or endometrialcancer, cancer of the oral cavity or pharynx, liver cancer, kidneycancer, testes cancer, biliary tract cancer, small bowel and appendixcancer, salivary gland cancer, thyroid gland cancer, adrenal glandcancer, and sarcomas such as osteosarcoma, chondrosarcoma, liposarcoma,and malignant fibrous histiocytoma. In general, MT-SP1 is a particularlygood marker for metastatic cancers.

E) Relevant Pathologies.

As indicated above, MT-SP1 provides an effective marker for detectionand/or evaluation of prognosis of a wide variety of cancers including,but not limited to, gastric cancer, prostate cancer, cancers of theurinary tract, lung cancer, bronchus cancer, a colorectal cancer (cancerof the colon and/or rectum), breast cancer, pancreas cancer, brain orcentral nervous system cancer, peripheral nervous system cancer,esophageal cancer, cervical cancer, melanoma, uterine or endometrialcancer, cancer of the oral cavity or pharynx, liver cancer, kidneycancer, testes cancer, biliary tract cancer, small bowel and appendixcancer, salivary gland cancer, thyroid gland cancer, adrenal glandcancer, and sarcomas such as osteosarcoma, chondrosarcoma, liposarcoma,and malignant fibrous histiocytoma. In general, MT-SP1 is a particularlygood marker for metastatic cancers.

IV. Assay Formats.

As indicated above, in one aspect, this invention is premised on thediscovery that membrane-type serine proteases (e.g. MT-SP1) areassociated with occurrence, growth, proliferation, invasiveness, andangiogenesis of cancers. Thus, in one embodiment, this inventionprovides methods of screening for cancers and/or evaluating the severityof a cancer and/or the likelihood of metastatic cells being presentand/or developing and/or evaluating the prognosis of a cancer. Themethods involve detecting the expression level and/or activity level ofa membrane-type serine protease where elevated expression levels and/orelevated activity levels indicate the presence of a cancer and/or thepresence of invasive cancer cells and/or increased severity of thedisease. Thus, assays of copy number, expression level or level ofactivity of one or MT-SP1 genes provides useful diagnostic and/orprognostic information. Using the nucleic acid sequences and/or aminoacid sequences provided herein copy number and/or activity level can bedirectly measured according to a number of different methods asdescribed below. In particular, expression levels of a gene can bealtered by changes in the copy number of the gene, and/or by changes inthe transcription of the gene product (i.e. transcription of mRNA),and/or by changes in translation of the gene product (i.e. translationof the protein), and/or by post-translational modification(s) (e.g.protein folding, glycosylation, etc.). Thus useful assays of thisinvention include assaying for copy number, level of transcribed mRNA,level of translated protein, activity of translated protein, etc.Examples of such approaches are described below.

A) Sample Collection and Processing.

The MT-SP1 nucleic acid and/or protein is preferably quantified in abiological sample derived from a mammal (e.g., whole blood, plasma,serum, synovial fluid, cerebrospinal fluid, bronchial lavage, ascitesfluid, bone marrow aspirate, pleural effusion, urine, or tumor tissue),more preferably from a human patient. Preferred biological samplesinlcude sample(s) of biological tissue or fluid that contain MT-SP1 in aconcentration that may be correlated with the presence and/or prognosisof a pathological state (e.g. a cancer). Particularly preferredbiological samples include, but are not limited to whole blood, serum,plasma, synovial fluid, cerebrospinal fluid, bronchial lavage, ascitesfluid, pleural effusion, bone marrow aspirate, urine, and tumor tissue.

The biological sample may be pretreated as necessary by dilution in anappropriate buffer solution or concentrated, if desired. Any of a numberof standard aqueous buffer solutions, employing one of a variety ofbuffers, such as phosphate, Tris, or the like, at physiological pH canbe used.

As indicated above, in a preferred embodiment, assays are performedusing whole blood, serum, or plasma or in tissue biopsies and/or tissuesections. Obtaining and storing tissues, blood and/or blood products arewell known to those of skill in the art. Typically blood is obtained byvenipuncture. The blood may be diluted by the addition of buffers orother reagents well known to those of skill in the art and may be storedfor up to 24 hours at 2-8° C., or at −20° C. or lower for longerperiods, prior to measurement of YKL-40. In a particularly preferredembodiment, the blood or blood product (e.g. serum) is stored at −70° C.without preservative indefinitely.

B) Nucleic-Acid Based Assays.

1) Target Molecules.

As indicated above, MT-SP1 gene expression can be varied by changes incopy number of the gene and/or changes in the regulation of geneexpression. Changes in copy number are most easily detected by directchanges in genomic DNA, while changes in expression level can bedetected by measuring changes in mRNA and/or a nucleic acid derived fromthe mRNA (e.g. reverse-transcribed cDNA, etc.).

In order to measure the nucleic acid concentration in a sample, it isdesirable to provide a nucleic acid sample for such analysis. Inpreferred embodiments the nucleic acid is found in or derived from abiological sample. The term “biological sample”, as used herein, refersto a sample obtained from an organism or from components (e.g., cells)of an organism. The sample may be of any biological tissue or fluid.Frequently the sample will be a “clinical sample” which is a samplederived from a patient. Such samples include, but are not limited to,sputum, blood, tissue or fine needle biopsy samples, urine, peritonealfluid, and pleural fluid, or cells therefrom. Biological samples mayalso include sections of tissues such as frozen sections taken forhistological purposes.

The nucleic acid (either genomic DNA or mRNA) is, in certain preferredembodiments, isolated from the sample according to any of a number ofmethods well known to those of skill in the art. One of skill willappreciate that where alterations in the copy number of a gene are to bedetected genomic DNA is preferably isolated. Conversely, whereexpression levels of a gene or genes are to be detected, preferably RNA(mRNA) is isolated.

Methods of isolating total mRNA are well known to those of skill in theart. For example, methods of isolation and purification of nucleic acidsare described in detail in by Tijssen ed., (1993) Chapter 3 ofLaboratory Techniques in Biochemistry and Molecular Biology:Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic AcidPreparation, Elsevier, N.Y. and Tijssen ed.

In a preferred embodiment, the “total” nucleic acid is isolated from agiven sample using, for example, an acid guanidinium-phenol-chloroformextraction method and polyA+ mRNA is isolated by oligo dT columnchromatography or by using (dT)n magnetic beads (see, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, ColdSpring Harbor Laboratory, (1989), or Current Protocols in MolecularBiology, F. Ausubel et al., ed. Greene Publishing andWiley-Interscience, New York (1987)).

Frequently, it is desirable to amplify the nucleic acid sample prior toassaying for gene copy number or expression level. One of skill in theart will appreciate that whatever amplification method is used, if aquantitative result is desired, care must be taken to use a method thatmaintains or controls for the relative frequencies of the amplifiednucleic acids.

Methods of “quantitative” amplification are well known to those of skillin the art. For example, quantitative PCR involves simultaneouslyco-amplifying a known quantity of a control sequence using the sameprimers. This provides an internal standard that may be used tocalibrate the PCR reaction.

One preferred internal standard is a synthetic AW106 cRNA. The AW106cRNA is combined with RNA isolated from the sample according to standardtechniques known to those of skill in the art. The RNA is then reversetranscribed using a reverse transcriptase to provide copy DNA. The cDNAsequences are then amplified (e.g., by PCR) using labeled primers. Theamplification products are separated, typically by electrophoresis, andthe amount of radioactivity (proportional to the amount of amplifiedproduct) is determined. The amount of mRNA in the sample is thencalculated by comparison with the signal produced by the known AW106 RNAstandard. Detailed protocols for quantitative PCR are provided in PCRProtocols, A Guide to Methods and Applications, Innis et al., AcademicPress, Inc. N.Y., (1990).

In a particularly preferred embodiment, where it is desired to quantifythe transcription level (and thereby expression) of a one or more genesin a sample, the nucleic acid sample is one in which the concentrationof the mRNA transcript(s) of the gene or genes, or the concentration ofthe nucleic acids derived from the mRNA transcript(s), is proportionalto the transcription level (and therefore expression level) of thatgene. Similarly, it is preferred that the hybridization signal intensitybe proportional to the amount of hybridized nucleic acid. While it ispreferred that the proportionality be relatively strict (e.g., adoubling in transcription rate results in a doubling in mRNA transcriptin the sample nucleic acid pool and a doubling in hybridization signal),one of skill will appreciate that the proportionality can be morerelaxed and even non-linear. Thus, for example, an assay where a 5 folddifference in concentration of the target mRNA results in a 3 to 6 folddifference in hybridization intensity is sufficient for most purposes.Where more precise quantification is required appropriate controls canbe run to correct for variations introduced in sample preparation andhybridization as described herein. In addition, serial dilutions of“standard” target nucleic acids (e.g., mRNAs) can be used to preparecalibration curves according to methods well known to those of skill inthe art. Of course, where simple detection of the presence or absence ofa transcript or large differences of changes in nucleic acidconcentration is desired, no elaborate control or calibration isrequired.

In the simplest embodiment, such a nucleic acid sample is the total mRNAor a total cDNA isolated and/or otherwise derived from a biologicalsample. The nucleic acid (either genomic DNA or mRNA) may be isolatedfrom the sample according to any of a number of methods well known tothose of skill in the art as indicated above.

2) Hybridization-Based Assays.

i) Detection of Copy Number.

One method for evaluating the copy number of an MT-SP1 DNA in a sampleinvolves a Southern transfer. In a Southern Blot, the DNA (e.g., genomicDNA), typically fragmented and separated on an electrophoretic gel, ishybridized to a probe specific for the target region. Comparison of theintensity of the hybridization signal from the probe for the targetregion with control probe signal from analysis of normal genomic DNA(e.g., a non-amplified portion of the same or related cell, tissue,organ, etc.) provides an estimate of the relative copy number of thetarget nucleic acid.

An alternative means for determining the copy number of an MT-SP1 geneof this invention is in situ hybridization. In situ hybridization assaysare well known (e.g., Angerer (1987) Meth. Enzymol 152: 649). Generally,in situ hybridization comprises the following major steps: (1) fixationof tissue or biological structure to be analyzed; (2) prehybridizationtreatment of the biological structure to increase accessibility oftarget DNA, and to reduce nonspecific binding; (3) hybridization of themixture of nucleic acids to the nucleic acid in the biological structureor tissue; (4) post-hybridization washes to remove nucleic acidfragments not bound in the hybridization and (5) detection of thehybridized nucleic acid fragments. The reagent used in each of thesesteps and the conditions for use vary depending on the particularapplication.

Preferred hybridization-based assays include, but are not limited to,traditional “direct probe” methods such as Southern blots or in situhybridization (e.g., FISH), and “comparative probe” methods such ascomparative genomic hybridization (CGH). The methods can be used in awide variety of formats including, but not limited to substrate—(e.g.membrane or glass) bound methods or array-based approaches as describedbelow.

In a typical in situ hybridization assay, cells are fixed to a solidsupport, typically a glass slide. If a nucleic acid is to be probed, thecells are typically denatured with heat or alkali. The cells are thencontacted with a hybridization solution at a moderate temperature topermit annealing of labeled probes specific to the nucleic acid sequenceencoding the protein. The targets (e.g., cells) are then typicallywashed at a predetermined stringency or at an increasing stringencyuntil an appropriate signal to noise ratio is obtained.

The probes are typically labeled, e.g., with radioisotopes orfluorescent reporters as described above. Preferred probes aresufficiently long so as to specifically hybridize with the targetnucleic acid(s) under stringent conditions. The preferred size range isfrom about 20 bases to about 500 bases, more preferably from about 30bases to about 400 bases and most preferably from about 40 bases toabout 300 bases.

In some applications it is necessary to block the hybridization capacityof repetitive sequences. Thus, in some embodiments, tRNA, human genomicDNA, or Cot-1 DNA is used to block non-specific hybridization.

Another effective approach for the quantification of copy number of thegene(s) or EST(s) of this invention is comparative genomichybridization. In this method, a first collection of (sample) nucleicacids (e.g. from a test sample derived from an organism, tissue, or cellexposed to one or more drugs of abuse) is labeled with a first label,while a second collection of (control) nucleic acids (e.g. from a normal“unexposed” organism, tissue, or cell) is labeled with a second label.The ratio of hybridization of the nucleic acids is determined by theratio of the two (first and second) labels binding to each fiber in thearray. Where there are chromosomal deletions or multiplications,differences in the ratio of the signals from the two labels will bedetected and the ratio will provide a measure of the gene and/or ESTcopy number.

Hybridization protocols suitable for use with the methods of theinvention are described, e.g., in Albertson (1984) EMBO J. 3: 1227-1234;Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No.430,402; Methods in Molecular Biology, Vol. 33: In Situ HybridizationProtocols, Choo, ed., Humana Press, Totowa, N.J. (1994), etc. In oneparticularly preferred embodiment, the hybridization protocol of Pinkelet al. (1998) Nature Genetics 20: 207-211, or of Kallioniemi (1992)Proc. Natl. Acad Sci USA 89:5321-5325 (1992) is used.

ii) Detection of Gene Transcript.

Methods of detecting and/or quantifying the transcript(s) of one or moreMT-SP1 gene(s) or EST(s) (e.g. mRNA or cDNA made therefrom) usingnucleic acid hybridization techniques are known to those of skill in theart (see Sambrook et al. supra). For example, one method for evaluatingthe presence, absence, or quantity of gene or EST reverse-transcribedcDNA involves a Southern transfer as described above. Alternatively, ina Northern blot, mRNA is directly quantitated. In brief, the mRNA isisolated from a given cell sample using, for example, an acidguanidinium-phenol-chloroform extraction method. The mRNA is thenelectrophoresed to separate the mRNA species and the mRNA is transferredfrom the gel to a nitrocellulose membrane. As with the Southern-blots,labeled probes are used to identify and/or quantify the target mRNA.

The probes used herein for detection of the MT-SP1 gene(s) and/or EST(s)of this invention can be full length or less than the full length of thegene or EST. Shorter probes are empirically tested for specificity.Preferably nucleic acid probes are 20 bases or longer in length. (seeSambrook et al. for methods of selecting nucleic acid probe sequencesfor use in nucleic acid hybridization.) Visualization of the hybridizedportions allows the qualitative determination of the presence or absenceof gene(s) and/or EST(s) of this invention.

3) Amplification-Based Assays.

In still another embodiment, amplification-based assays can be used tomeasure or level of gene (or EST) transcript. In suchamplification-based assays, the target nucleic acid sequences act astemplate(s) in amplification reaction(s) (e.g. Polymerase Chain Reaction(PCR) or reverse-transcription PCR (RT-PCR)). In a quantitativeamplification, the amount of amplification product will be proportionalto the amount of template in the original sample. Comparison toappropriate (e.g. healthy tissue unexposed to drug(s) of abuse) controlsprovides a measure of the copy number or transcript level of the targetgene or EST.

Methods of “quantitative” amplification are well known to those of skillin the art. For example, quantitative PCR involves simultaneouslyco-amplifying a known quantity of a control sequence using the sameprimers. This provides an internal standard that may be used tocalibrate the PCR reaction. Detailed protocols for quantitative PCR areprovided in Innis et al. (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc. N.Y.). The known nucleic acidsequence(s) for the cDNA, genes, and ESTs provided herein is sufficientto enable one of skill to routinely select primers to amplify anyportion of the gene.

Other suitable amplification methods include, but are not limited toligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560,Landegren et al. (1988) Science 241: 1077, and Barringer et al. (1990)Gene 89: 117, transcription amplification (Kwoh et al. (1989) Proc.Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication(Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR,and linker adapter PCR, etc.

As indicated above, PCR assay methods are well known to those of skillin the art. Similarly, RT-PCR methods are also well known.

4) Hybridization Formats and Optimization of Hybridization Conditions.

i) Array-Based Hybridization Formats.

In one embodiment, the methods of this invention can be utilized inarray-based hybridization formats. Arrays are a multiplicity ofdifferent “probe” or “target” nucleic acids (or other compounds)attached to one or more surfaces (e.g., solid, membrane, or gel). In apreferred embodiment, the multiplicity of nucleic acids (or othermoieties) is attached to a single contiguous surface or to amultiplicity of surfaces juxtaposed to each other.

In an array format a large number of different hybridization reactionscan be run essentially “in parallel.” This provides rapid, essentiallysimultaneous, evaluation of a number of hybridizations in a single“experiment”. Methods of performing hybridization reactions in arraybased formats are well known to those of skill in the art (see, e.g.,Pastinen (1997) Genome Res. 7: 606-614; Jackson (1996) NatureBiotechnology 14:1685; Chee (1995) Science 274: 610; WO 96/17958, Pinkelet al. (1998) Nature Genetics 20: 207-211).

Arrays, particularly nucleic acid arrays can be produced according to awide variety of methods well known to those of skill in the art. Forexample, in a simple embodiment, “low density” arrays can simply beproduced by spotting (e.g. by hand using a pipette) different nucleicacids at different locations on a solid support (e.g. a glass surface, amembrane, etc.).

This simple spotting, approach has been automated to produce highdensity spotted arrays (see, e.g., U.S. Pat. No. 5,807,522). This patentdescribes the use of an automated system that taps a microcapillaryagainst a surface to deposit a small volume of a biological sample. Theprocess is repeated to generate high density arrays.

Arrays can also be produced using oligonucleotide synthesis technology.Thus, for example, U.S. Pat. No. 5,143,854 and PCT Patent PublicationNos. WO 90/15070 and 92/10092 teach the use of light-directedcombinatorial synthesis of high density oligonucleotide arrays.Synthesis of high density arrays is also described in U.S. Pat. Nos.5,744,305, 5,800,992 and 5,445,934.

In a preferred embodiment, the arrays used in this invention comprise“probe” nucleic acids. These probes or target nucleic acids are thenhybridized respectively with their “target” nucleic acids (e.g., mRNAderived from a biological sample).

In another embodiment the array, particularly a spotted array, caninclude genomic DNA, e.g. one or more clones that provide a highresolution scan of the genome containing the gene(s) and/or EST(s) ofthis invention. The nucleic acid clones can be obtained from, e.g.,HACs, MACs, YACs, BACs, PACs, P1s, cosmids, plasmids, inter-Alu PCRproducts of genomic clones, restriction digests of genomic clones, cDNAclones, amplification (e.g., PCR) products, and the like.

In various embodiments, the array nucleic acids are derived frompreviously mapped libraries of clones spanning or including thesequences of the invention. The arrays can be hybridized with a singlepopulation of sample nucleic acid or can be used with two differentiallylabeled collections (as with a test sample and a reference sample).

Many methods for immobilizing nucleic acids on a variety of solidsurfaces are known in the art. A wide variety of organic and inorganicpolymers, as well as other materials, both natural and synthetic, can beemployed as the material for the solid surface. Illustrative solidsurfaces include, e.g., nitrocellulose, nylon, glass, quartz, diazotizedmembranes (paper or nylon), silicones, polyformaldehyde, cellulose, andcellulose acetate. In addition, plastics such as polyethylene,polypropylene, polystyrene, and the like can be used. Other materialswhich may be employed include paper, ceramics, metals, metalloids,semiconductive materials, cermets or the like. In addition, substancesthat form gels can be used. Such materials include, e.g., proteins(e.g., gelatins), lipopolysaccharides, silicates, agarose andpolyacrylamides. Where the solid surface is porous, various pore sizesmay be employed depending upon the nature of the system.

In preparing the surface, a plurality of different materials may beemployed, particularly as laminates, to obtain various properties. Forexample, proteins (e.g., bovine serum albumin) or mixtures ofmacromolecules (e.g., Denhardt's solution) can be employed to avoidnon-specific binding, simplify covalent conjugation, enhance signaldetection or the like. If covalent bonding between a compound and thesurface is desired, the surface will usually be polyfunctional or becapable of being polyfunctionalized. Functional groups which may bepresent on the surface and used for linking can include carboxylicacids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxylgroups, mercapto groups and the like. The manner of linking a widevariety of compounds to various surfaces is well known and is amplyillustrated in the literature.

For example, methods for immobilizing nucleic acids by introduction ofvarious functional groups to the molecules is known (see, e.g., Bischoff(1987) Anal. Biochem., 164: 336-344; Kremsky (1987) Nucl. Acids Res. 15:2891-2910). Modified nucleotides can be placed on the target using PCRprimers containing the modified nucleotide, or by enzymatic end labelingwith modified nucleotides. Use of glass or membrane supports (e.g.,nitrocellulose, nylon, polypropylene) for the nucleic acid arrays of theinvention is advantageous because of well developed technology employingmanual and robotic methods of arraying targets at relatively highelement densities. Such membranes are generally available and protocolsand equipment for hybridization to membranes is well known.

Target elements of various sizes, ranging from 1 mm diameter down to 1μm can be used. Relatively simple approaches capable of quantitativefluorescent imaging of 1 cm² areas have been described that permitacquisition of data from a large number of target elements in a singleimage (see, e.g., Wittrup (1994) Cytometry 16:206-213, Pinkel et al.(1998) Nature Genetics 20: 207-211).

Arrays on solid surface substrates with much lower fluorescence thanmembranes, such as glass, quartz, or small beads, can achieve muchbetter sensitivity. Substrates such as glass or fused silica areadvantageous in that they provide a very low fluorescence substrate, anda highly efficient hybridization environment. Covalent attachment of thetarget nucleic acids to glass or synthetic fused silica can beaccomplished according to a number of known techniques (describedabove). Nucleic acids can be conveniently coupled to glass usingcommercially available reagents. For instance, materials for preparationof silanized glass with a number of functional groups are commerciallyavailable or can be prepared using standard techniques (see, e.g., Gait(1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press,Wash., D.C.). Quartz cover slips, which have at least 10-fold lowerautofluorescence than glass, can also be silanized.

Alternatively, probes can also be immobilized on commercially availablecoated beads or other surfaces. For instance, biotin end-labeled nucleicacids can be bound to commercially available avidin-coated beads.Streptavidin or anti-digoxigenin antibody can also be attached tosilanized glass slides by protein-mediated coupling using e.g., proteinA following standard protocols (see, e.g., Smith (1992) Science 258:1122-1126). Biotin or digoxigenin end-labeled nucleic acids can beprepared according to standard techniques. Hybridization to nucleicacids attached to beads is accomplished by suspending them in thehybridization mix, and then depositing them on the glass substrate foranalysis after washing. Alternatively, paramagnetic particles, such asferric oxide particles, with or without avidin coating, can be used.

ii) Other Hybridization Formats.

A variety of nucleic acid hybridization formats are known to thoseskilled in the art. For example, common formats include sandwich assaysand competition or displacement assays. Hybridization techniques aregenerally described in Hames and Higgins (1985) Nucleic AcidHybridization, A Practical Approach, IRL Press; Gall and Pardue (1969)Proc. Natl. Acad. Sci. USA 63: 378-383; and John et al. (1969) Nature223: 582-587.

Sandwich assays are commercially useful hybridization assays fordetecting or isolating nucleic acid sequences. Such assays utilize a“capture” nucleic acid covalently immobilized to a solid support and alabeled “signal” nucleic acid in solution. The sample will provide thetarget nucleic acid. The “capture” nucleic acid and “signal” nucleicacid probe hybridize with the target nucleic acid to form a “sandwich”hybridization complex. To be most effective, the signal nucleic acidshould not hybridize with the capture nucleic acid.

Typically, labeled signal nucleic acids are used to detecthybridization. Complementary nucleic acids or signal nucleic acids maybe labeled by any one of several methods typically used to detect thepresence of hybridized polynucleotides. The most common method ofdetection is the use of autoradiography with ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²Plabelled probes or the like. Other labels include ligands that bind tolabeled antibodies, fluorophores, chemi-luminescent agents, enzymes, andantibodies which can serve as specific binding pair members for alabeled ligand.

Detection of a hybridization complex may require the binding of a signalgenerating complex to a duplex of target and probe polynucleotides ornucleic acids. Typically, such binding occurs through ligand andanti-ligand interactions as between a ligand-conjugated probe and ananti-ligand conjugated with a signal.

The sensitivity of the hybridization assays may be enhanced through useof a nucleic acid amplification system that multiplies the targetnucleic acid being detected. Examples of such systems include thepolymerase chain reaction (PCR) system and the ligase chain reaction(LCR) system. Other methods recently described in the art are thenucleic acid sequence based amplification (NASBAO, Cangene, Mississauga,Ontario) and Q Beta Replicase systems.

iii) Optimization of Hybridization Conditions.

Nucleic acid hybridization simply involves providing a denatured probeand target nucleic acid under conditions where the probe and itscomplementary target can form stable hybrid duplexes throughcomplementary base pairing. The nucleic acids that do not form hybridduplexes are then washed away leaving the hybridized nucleic acids to bedetected, typically through detection of an attached detectable label.It is generally recognized that nucleic acids are denatured byincreasing the temperature or decreasing the salt concentration of thebuffer containing the nucleic acids, or in the addition of chemicalagents, or the raising of the pH. Under low stringency conditions (e.g.,low temperature and/or high salt and/or high target concentration)hybrid duplexes (e.g., DNA:DNA, RNA:RNA, or RNA:DNA) will form evenwhere the annealed sequences are not perfectly complementary. Thusspecificity of hybridization is reduced at lower stringency. Conversely,at higher stringency (e.g., higher temperature or lower salt) successfulhybridization requires fewer mismatches.

One of skill in the art will appreciate that hybridization conditionsmay be selected to provide any degree of stringency. In a preferredembodiment, hybridization is performed at low stringency to ensurehybridization and then subsequent washes are performed at higherstringency to eliminate mismatched hybrid duplexes. Successive washesmay be performed at increasingly higher stringency (e.g., down to as lowas 0.25×SSPE at 37° C. to 70° C.) until a desired level of hybridizationspecificity is obtained. Stringency can also be increased by addition ofagents such as formamide. Hybridization specificity may be evaluated bycomparison of hybridization to the test probes with hybridization to thevarious controls that can be present.

In general, there is a tradeoff between hybridization specificity(stringency) and signal intensity. Thus, in a preferred embodiment, thewash is performed at the highest stringency that produces consistentresults and that provides a signal intensity greater than approximately10% of the background intensity. Thus, in a preferred embodiment, thehybridized array may be washed at successively higher stringencysolutions and read between each wash. Analysis of the data sets thusproduced will reveal a wash stringency above which the hybridizationpattern is not appreciably altered and which provides adequate signalfor the particular probes of interest.

In a preferred embodiment, background signal is reduced by the use of ablocking reagent (e.g., tRNA, sperm DNA, cot-1 DNA, etc.) during thehybridization to reduce non-specific binding. The use of blocking agentsin hybridization is well known to those of skill in the art (see, e.g.,Chapter 8 in P. Tijssen, supra.)

Methods of optimizing hybridization conditions are well known to thoseof skill in the art (see, e.g., Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 24: Hybridization With NucleicAcid Probes, Elsevier, N.Y.).

Optimal conditions are also a function of the sensitivity of label(e.g., fluorescence) detection for different combinations of substratetype, fluorochrome, excitation and emission bands, spot size and thelike. Low fluorescence background surfaces can be used (see, e.g., Chu(1992) Electrophoresis 13:105-114). The sensitivity for detection ofspots (“target elements”) of various diameters on the candidate surfacescan be readily determined by, e.g., spotting a dilution series offluorescently end labeled DNA fragments. These spots are then imagedusing conventional fluorescence microscopy. The sensitivity, linearity,and dynamic range achievable from the various combinations offluorochrome and solid surfaces (e.g., glass, fused silica, etc.) canthus be determined. Serial dilutions of pairs of fluorochrome in knownrelative proportions can also be analyzed. This determines the accuracywith which fluorescence ratio measurements reflect actual fluorochromeratios over the dynamic range permitted by the detectors andfluorescence of the substrate upon which the probe has been fixed.

iv) Labeling and Detection of Nucleic Acids.

In a preferred embodiment, the hybridized nucleic acids are detected bydetecting one or more labels attached to the sample nucleic acids. Thelabels may be incorporated by any of a number of means well known tothose of skill in the art. Means of attaching labels to nucleic acidsinclude, for example nick translation, or end-labeling by kinasing ofthe nucleic acid and subsequent attachment (ligation) of a linkerjoining the sample nucleic acid to a label (e.g., a fluorophore). A widevariety of linkers for the attachment of labels to nucleic acids arealso known. In addition, intercalating dyes and fluorescent nucleotidescan also be used.

Detectable labels suitable for use in the present invention include anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include biotin for staining with labeledstreptavidin conjugate, magnetic beads (e.g., Dynabeads™), fluorescentdyes (e.g., fluorescein, texas red, rhodamine, green fluorescentprotein, and the like, see, e.g., Molecular Probes, Eugene, Oreg., USA),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in anELISA), and colorimetric labels such as colloidal gold (e.g., goldparticles in the 40-80 nm diameter size range scatter green light withhigh efficiency) or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads. Patents teaching the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241.

A fluorescent label is preferred because it provides a very strongsignal with low background. It is also optically detectable at highresolution and sensitivity through a quick scanning procedure. Thenucleic acid samples can all be labeled with a single label, e.g., asingle fluorescent label. Alternatively, in another embodiment,different nucleic acid samples can be simultaneously hybridized whereeach nucleic acid sample has a different label. For instance, one targetcould have a green fluorescent label and a second target could have ared fluorescent label. The scanning step will distinguish sites ofbinding of the red label from those binding the green fluorescent label.Each nucleic acid sample (target nucleic acid) can be analyzedindependently from one another.

Suitable chromogens which can be employed include those molecules andcompounds which absorb light in a distinctive range of wavelengths sothat a color can be observed or, alternatively, which emit light whenirradiated with radiation of a particular wave length or wave lengthrange, e.g., fluorescers.

Desirably, fluorescers should absorb light above about 300 nm,preferably about 350 nm, and more preferably above about 400 nm, usuallyemitting at wavelengths greater than about 10 nm higher than thewavelength of the light absorbed. It should be noted that the absorptionand emission characteristics of the bound dye can differ from theunbound dye. Therefore, when referring to the various wavelength rangesand characteristics of the dyes, it is intended to indicate the dyes asemployed and not the dye which is unconjugated and characterized in anarbitrary solvent.

Fluorescers are generally preferred because by irradiating a fluorescerwith light, one can obtain a plurality of emissions. Thus, a singlelabel can provide for a plurality of measurable events.

Detectable signal can also be provided by chemiluminescent andbioluminescent sources. Chemiluminescent sources include a compoundwhich becomes electronically excited by a chemical reaction and can thenemit light which serves as the detectable signal or donates energy to afluorescent acceptor. Alternatively, luciferins can be used inconjunction with luciferase or lucigenins to provide bioluminescence.

Spin labels are provided by reporter molecules with an unpaired electronspin which can be detected by electron spin resonance (ESR)spectroscopy. Exemplary spin labels include organic free radicals,transitional metal complexes, particularly vanadium, copper, iron, andmanganese, and the like. Exemplary spin labels include nitroxide freeradicals.

The label may be added to the target (sample) nucleic acid(s) prior to,or after the hybridization. So called “direct labels” are detectablelabels that are directly attached to or incorporated into the target(sample) nucleic acid prior to hybridization. In contrast, so called“indirect labels” are joined to the hybrid duplex after hybridization.Often, the indirect label is attached to a binding moiety that has beenattached to the target nucleic acid prior to the hybridization. Thus,for example, the target nucleic acid may be biotinylated before thehybridization. After hybridization, an avidin-conjugated fluorophorewill bind the biotin bearing hybrid duplexes providing a label that iseasily detected. For a detailed review of methods of labeling nucleicacids and detecting labeled hybridized nucleic acids see LaboratoryTechniques in Biochemistry and Molecular Biology, Vol. 24: HybridizationWith Nucleic Acid Probes, P. Tijssen, ed. Elsevier, N.Y., (1993)).

Fluorescent labels are easily added during an in vitro transcriptionreaction. Thus, for example, fluorescein labeled UTP and CTP can beincorporated into the RNA produced in an in vitro transcription.

The labels can be attached directly or through a linker moiety. Ingeneral, the site of label or linker-label attachment is not limited toany specific position. For example, a label may be attached to anucleoside, nucleotide, or analogue thereof at any position that doesnot interfere with detection or hybridization as desired. For example,certain Label-ON Reagents from Clontech (Palo Alto, Calif.) provide forlabeling interspersed throughout the phosphate backbone of anoligonucleotide and for terminal labeling at the 3′ and 5′ ends. Asshown for example herein, labels can be attached at positions on theribose ring or the ribose can be modified and even eliminated asdesired. The base moieties of useful labeling reagents can include thosethat are naturally occurring or modified in a manner that does notinterfere with the purpose to which they are put. Modified bases includebut are not limited to 7-deaza A and G, 7-deaza-8-aza A and G, and otherheterocyclic moieties.

It will be recognized that fluorescent labels are not to be limited tosingle species organic molecules, but include inorganic molecules,multi-molecular mixtures of organic and/or inorganic molecules,crystals, heteropolymers, and the like. Thus, for example, CdSe—CdScore-shell nanocrystals enclosed in a silica shell can be easilyderivatized for coupling to a biological molecule (Bruchez et al. (1998)Science, 281: 2013-2016). Similarly, highly fluorescent quantum dots(zinc sulfide-capped cadmium selenide) have been covalently coupled tobiomolecules for use in ultrasensitive biological detection (Warren andNie (1998) Science, 281: 2016-2018).

C) Polypeptide-Based Assays.

In addition to, or in alternative to, the detection of nucleic acidlevel(s), alterations in expression of MT-SP1 can be detected and/orquantified by detecting and/or quantifying the amount and/or activity oftranslated MT-SP1 protein(s). In particularly preferred embodiments, theMT-SP1 proteins are detected immunohistochemically, using aradioimmunoassay, or using other immunoassay(s). As used herein, animmunoassay is an assay that utilizes an antibody to specifically bindto the analyte (MT-SP1). The immunoassay is thus characterized bydetection of specific binding of a MT-SP1 protein, or protein fragment,to an anti-MT-SP1 antibody as opposed to the use of other physical orchemical properties to isolate, target, and quantify the analyte.

1) Detection of Expressed Protein

The MT-SP1 polypeptide(s) of this invention can be detected andquantified by any of a number of methods well known to those of skill inthe art. These may include analytic biochemical methods such aselectrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), hyperdiffusionchromatography, and the like, or various immunological methods such asfluid or gel precipitin reactions, immunodiffusion (single or double),immunohistochemistry, affinity chromatography, immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, western blotting, and the like.

In one preferred embodiment, the MT-SP1 polypeptide(s) are detectedand/or quantified using immunohistochemical methods. In this approach,antibodies that specifically bind to an MT-SP1 are contacted with thebiological sample (e.g., a histological sample). Those antibodies thatspecifically bind to the sample are visualized, or otherwise detected,and provide an indication of the location, presence, absence or quantityof MT-SP1 protein in the sample. The antibodies are typically detectedby detection of a label either affixed to the antibody prior to orsubsequent to the “contacting” step. Immunohistochemical methods arewell known to those of skill in the art (see, e.g., Kleihues et al.(1993) Histological typing of tumours of the central nervous system,Springer Verlag, New York).

In another preferred embodiment, the MT-SP1 polypeptide(s) aredetected/quantified in an electrophoretic protein separation (e.g. a 1-or 2-dimensional electrophoresis). Means of detecting proteins usingelectrophoretic techniques are well known to those of skill in the art(see generally, R. Scopes (1982) Protein Purification, Springer-Verlag,N.Y.; Deutscher, (1990) Methods in Enzymology Vol. 182: Guide to ProteinPurification, Academic Press, Inc., N.Y.).

Another preferred embodiment utilizes a Western blot (immunoblot)analysis to detect and quantify the presence of polypeptide(s) of thisinvention in the sample. This technique generally comprises separatingsample proteins by gel electrophoresis on the basis of molecular weight,transferring the separated proteins to a suitable solid support, (suchas a nitrocellulose filter, a nylon filter, or derivatized nylonfilter), and incubating the sample with the antibodies that specificallybind the target polypeptide(s).

The antibodies specifically bind to the target polypeptide(s) and may bedirectly labeled or alternatively may be subsequently detected usinglabeled antibodies (e.g., labeled sheep anti-mouse antibodies) thatspecifically bind to the a domain of the antibody.

Other suitable assay formats include, but are not limited to, liposomeimmunoassays (LIA), which use liposomes designed to bind specificmolecules (e.g., antibodies) and release encapsulated reagents ormarkers. The released chemicals are then detected according to standardtechniques (see, Monroe et al. (1986) Amer. Clin. Prod. Rev. 5: 34-41).

In a preferred embodiment, the MT-SP1 protein(s) are detected and/orquantified in the biological sample using any of a number of wellrecognized immunological binding assays (immunoassays) (see, e.g., U.S.Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a reviewof the general immunoassays, see also Methods in Cell Biology Volume 37:Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York(1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, eds.(1991).

As used herein, an immunoassay is an assay that utilizes an antibody tospecifically bind to the analyte (e.g., an MT-SP1 polypeptide). Theimmunoassay is thus characterized by detection of specific binding of apolypeptide of this invention to an antibody as opposed to the use ofother physical or chemical properties to isolate, target, and quantifythe analyte.

Immunological binding assays (or immunoassays) typically utilize a“capture agent” to specifically bind to and often immobilize the analyte(in this case an MT-SP1 polypeptide). In preferred embodiments, thecapture agent is an antibody.

Immunoassays also often utilize a labeling agent to specifically bind toand label the binding complex formed by the capture agent and theanalyte. The labeling agent may itself be one of the moieties comprisingthe antibody/analyte complex. Thus, the labeling agent may be a labeledpolypeptide or a labeled antibody that specifically recognizes thealready bound target polypeptide. Alternatively, the labeling agent maybe a third moiety, such as another antibody, that specifically binds tothe capture agent/polypeptide complex.

Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally Kronval, et al. (1973) J. Immunol., 111: 1401-1406, andAkerstrom (1985) J. Immunol., 135: 2589-2542).

As indicated above, immunoassays for the detection and/or quantificationof the MT-SP1 polypeptide(s) of this invention can take a wide varietyof formats well known to those of skill in the art. Preferredimmunoassays for detecting the target polypeptide(s) are eithercompetitive or noncompetitive. Noncompetitive immunoassays are assays inwhich the amount of captured analyte is directly measured. In onepreferred “sandwich” assay, for example, the capture agents (antibodies)can be bound directly to a solid substrate where they are immobilized.These immobilized antibodies then capture the target polypeptide presentin the test sample. The target polypeptide thus immobilized is thenbound by a labeling agent, such as a second antibody bearing a label.

In competitive assays, the amount of analyte (MT-SP1 polypeptide)present in the sample is measured indirectly by measuring the amount ofan added (exogenous) analyte displaced (or competed away) from a captureagent (antibody) by the analyte present in the sample. In onecompetitive assay, a known amount of, in this case, labeled polypeptideis added to the sample and the sample is then contacted with a captureagent. The amount of labeled polypeptide bound to the antibody isinversely proportional to the concentration of target polypeptidepresent in the sample.

In one particularly preferred embodiment, the antibody is immobilized ona solid substrate. The immobilized antibody captures the target MT-SP1thereby immobilizing the analyte. The amount of analyte (targetpolypeptide) bound to the antibody may be determined either by measuringthe amount of target polypeptide present in the polypeptide/antibodycomplex, or alternatively by measuring the amount of remaininguncomplexed polypeptide.

The assays of this invention are scored (as positive or negative orquantity of target polypeptide) according to standard methods well knownto those of skill in the art. The particular method of scoring willdepend on the assay format and choice of label. For example, a WesternBlot assay can be scored by visualizing the colored product produced bythe enzymatic label. A clearly visible colored band or spot at thecorrect molecular weight is scored as a positive result, while theabsence of a clearly visible spot or band is scored as a negative. Theintensity of the band or spot can provide a quantitative measure oftarget polypeptide concentration.

Antibodies for use in the various immunoassays described herein, can beproduced as described below.

2) Detection of Enzyme Activity.

In another embodiment, levels of gene expression/regulation are assayedby measuring the enzymatic activity of the polypeptide encoded by therespective gene(s). For example, the MT-SP1 polypeptide(s) of thisinvention are serine proteases and their activity can be readilydetected by assaying the cleavage of a target substrate. Thus, Example 1illustrated quantification of MT-SP1 activity using an active sitetitration with MUGB. The catalytic activity of the protease domain canalso be monitored using pNA substrates. In particular, MT-SP1 proteaseactivity can be tested against tetrapeptide substrates of the formSuc-AAPX-pNA, which contained various amino acids at the P1 position(P1-Ala, Asp, Glu, Phe, Leu, Met, Lys, or Arg). In a preferredembodiment, substrates with P1-Lys or P1-Arg are used. Protease domaincan also be characterized using the substrate Spectrozyme tPA(hexahydrotyrosyl-Gly-Arg-pNA) as described in Example 1. Using theteaching provided herein, assays for activity of other MT-SP1 proteasesare easily performed.

3) Antibodies to Polypeptides Expressed by the Genes or ESTs IdentifiedHerein.

Either polyclonal or monoclonal antibodies may be used in theimmunoassays of the invention described herein. Polyclonal antibodiesare preferably raised by multiple injections (e.g. subcutaneous orintramuscular injections) of substantially pure polypeptides orantigenic polypeptides into a suitable non-human mammal. Theantigenicity of the target peptides can be determined by conventionaltechniques to determine the magnitude of the antibody response of ananimal that has been immunized with the peptide. Generally, the peptidesthat are used to raise antibodies for use in the methods of thisinvention should generally be those which induce production of hightiters of antibody with relatively high affinity for target polypeptidesencoded by the MT-SP1 genes or ESTs of this invention.

If desired, the immunizing peptide may be coupled to a carrier proteinby conjugation using techniques that are well-known in the art. Suchcommonly used carriers which are chemically coupled to the peptideinclude keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serumalbumin (BSA), and tetanus toxoid. The coupled peptide is then used toimmunize the animal (e.g. a mouse or a rabbit).

The antibodies are then obtained from blood samples taken from themammal. The techniques used to develop polyclonal antibodies are knownin the art (see, e.g., Methods of Enzymology, “Production of AntiseraWith Small Doses of Immunogen: Multiple Intradermal Injections”,Langone, et al. eds. (Acad. Press, 1981)). Polyclonal antibodiesproduced by the animals can be further purified, for example, by bindingto and elution from a matrix to which the peptide to which theantibodies were raised is bound. Those of skill in the art will know ofvarious techniques common in the immunology arts for purification and/orconcentration of polyclonal antibodies, as well as monoclonal antibodiessee, for example, Coligan, et al. (1991) Unit 9, Current Protocols inImmunology, Wiley Interscience).

Preferably, however, the antibodies produced will be monoclonalantibodies (“mAb's”). For preparation of monoclonal antibodies,immunization of a mouse or rat is preferred. The term “antibody” as usedin this invention includes intact molecules as well as fragmentsthereof, such as, Fab and F(ab′)^(2′), and/or single-chain antibodies(e.g. scFv) which are capable of binding an epitopic determinant. Also,in this context, the term “mab's of the invention” refers to monoclonalantibodies with specificity for a polypeptide encoded by an MT-SP1 geneor EST.

The general method used for production of hybridomas secreting mAbs iswell known (Kohler and Milstein (1975) Nature, 256:495). Briefly, asdescribed by Kohler and Milstein the technique comprised isolatinglymphocytes from regional draining lymph nodes of five separate cancerpatients with either melanoma, teratocarcinoma or cancer of the cervix,glioma or lung, (where samples were obtained from surgical specimens),pooling the cells, and fusing the cells with SHFP-1. Hybridomas werescreened for production of antibody which bound to cancer cell lines.Confirmation of specificity among mAb's can be accomplished usingrelatively routine screening techniques (such as the enzyme-linkedimmunosorbent assay, or “ELISA”) to determine the elementary reactionpattern of the mAb of interest.

It is also possible to evaluate an mAb to determine whether it has thesame specificity as a mAb of the invention without undue experimentationby determining whether the mAb being tested prevents a mAb of theinvention from binding to the target polypeptide isolated as describedabove. If the mAb being tested competes with the mAb of the invention,as shown by a decrease in binding by the mAb of the invention, then itis likely that the two monoclonal antibodies bind to the same or aclosely related epitope. Still another way to determine whether a mAbhas the specificity of a mAb of the invention is to preincubate the mAbof the invention with an antigen with which it is normally reactive, anddetermine if the mAb being tested is inhibited in its ability to bindthe antigen. If the mAb being tested is inhibited then, in alllikelihood, it has the same, or a closely related, epitopic specificityas the mAb of the invention.

Antibodies fragments, e.g. single chain antibodies (scFv or others), canalso be produced/selected using phage display technology. The ability toexpress antibody fragments on the surface of viruses that infectbacteria (bacteriophage or phage) makes it possible to isolate a singlebinding antibody fragment, e.g., from a library of greater than 10¹⁰nonbinding clones. To express antibody fragments on the surface of phage(phage display), an antibody fragment gene is inserted into the geneencoding a phage surface protein (e.g., pill) and the antibodyfragment-pIII fusion protein is displayed on the phage surface(McCafferty et al. (1990) Nature, 348: 552-554; Hoogenboom et al. (1991)Nucleic Acids Res. 19: 4133-4137).

Since the antibody fragments on the surface of the phage are functional,phage bearing antigen binding antibody fragments can be separated fromnon-binding phage by antigen affinity chromatography (McCafferty et al.(1990) Nature, 348: 552-554). Depending on the affinity of the antibodyfragment, enrichment factors of 20 fold-1,000,000 fold are obtained fora single round of affinity selection. By infecting bacteria with theeluted phage, however, more phage can be grown and subjected to anotherround of selection. In this way, an enrichment of 1000 fold in one roundcan become 1,000,000 fold in two rounds of selection (McCafferty et al.(1990) Nature, 348: 552-554). Thus even when enrichments are low (Markset al. (1991) J. Mol. Biol. 222: 581-597), multiple rounds of affinityselection can lead to the isolation of rare phage. Since selection ofthe phage antibody library on antigen results in enrichment, themajority of clones bind antigen after as few as three to four rounds ofselection. Thus only a relatively small number of clones (severalhundred) need to be analyzed for binding to antigen.

Human antibodies can be produced without prior immunization bydisplaying very large and diverse V-gene repertoires on phage (Marks etal. (1991) J. Mol. Biol. 222: 581-597). In one embodiment natural V_(H)and V_(L) repertoires present in human peripheral blood lymphocytes arewere isolated from unimmunized donors by PCR. The V-gene repertoireswere spliced together at random using PCR to create a scFv generepertoire which is was cloned into a phage vector to create a libraryof 30 million phage antibodies (Id.). From this single “naive” phageantibody library, binding antibody fragments have been isolated againstmore than 17 different antigens, including haptens, polysaccharides andproteins (Marks et al. (1991) J. Mol. Biol. 222: 581-597; Marks et al.(1993). Bio/Technology. 10: 779-783; Griffiths et al. (1993) EMBO J. 12:725-734; Clackson et al. (1991) Nature. 352: 624-628). Antibodies havebeen produced against self proteins, including human thyroglobulin,immunoglobulin, tumor necrosis factor and CEA (Griffiths et al. (1993)EMBO J. 12: 725-734). It is also possible to isolate antibodies againstcell surface antigens by selecting directly on intact cells. Theantibody fragments are highly specific for the antigen used forselection and have affinities in the 1 μM to 100 nM range (Marks et al.(1991) J. Mol. Biol. 222: 581-597; Griffiths et al. (1993) EMBO J. 12:725-734). Larger phage antibody libraries result in the isolation ofmore antibodies of higher binding affinity to a greater proportion ofantigens.

It will also be recognized that antibodies can be prepared by any of anumber of commercial services (e.g., Berkeley antibody laboratories,Bethyl Laboratories, Anawa, Eurogenetec, etc.).

V. MT-SP1 as a Target for Screening for Therapeutics.

A) Screening Target for Agents that Modulate MT-SP1 Expression and/orActivity.

While, in one embodiment, the assays described above provided methods ofdetecting the presence or absence, or quantifying expression of anMT-SP1 protease, it will be appreciated that the same assays can be usedto screen for agents that modulate the expression of and/or the activityof an MT-SP1 serine protease. To screen for potential modulators, theassays described above are performed in the presence of one or more testagents or are performed using biological samples from cells and/ortissues and/or organs and/or organisms exposed to one or more testagents. The MT-SP1 activity and/or expression level is determined and,in a preferred embodiment, compared to the activity level(s) observed in“control” assays (e.g., the same assays lacking the test agent). Adifference between the MT-SP1 expression and/or activity in the “test”assay as compared to the control assay indicates that the test agent isa “modulator” of SP1 expression and/or activity.

In a preferred embodiment, the assays of this invention level are deemedto show a positive result, e.g. elevated expression and/or MT-SP1activity, genes, when the measured protein or nucleic acid level orprotein activity is greater than the level measured or known for acontrol sample (e.g. either a level known or measured for a normalhealthy cell, tissue or organism mammal of the same species not exposedto the or putative modulator (test agent), or a “baseline/reference”level determined at a different tissue and/or a different time for thesame individual). In a particularly preferred embodiment, the assay isdeemed to show a positive result when the difference between sample and“control” is statistically significant (e.g. at the 85% or greater,preferably at the 90% or greater, more preferably at the 95% or greaterand most preferably at the 98% or greater confidence level).

B) Pre-Screening for Agents that Specifically Bind/Interact with MT-SP1.

In certain embodiments it is desired to pre-screen test agents for theability to interact with (e.g. specifically bind to) a MT-SP1 nucleicacid or polypeptide. Specifically binding test agents are more likely tointeract with and thereby modulate MT-SP1 expression and/or activity.Thus, in some preferred embodiments, the test agent(s) are pre-screenedfor binding to MT-SP1 or to an MT-SP1 nucleic acid before performing themore complex assays described above.

In one embodiment, such pre-screening is accomplished with simplebinding assays. Means of assaying for specific binding or the bindingaffinity of a particular ligand for a nucleic acid or for a protein arewell known to those of skill in the art. In preferred binding assays,the MT-SP1 protein or nucleic acid is immobilized and exposed to a testagent (which can be labeled), or alternatively, the test agent(s) areimmobilized and exposed to an MT-SP1 or to a MT-SP1 nucleic acid (whichcan be labeled). The immobilized moiety is then washed to remove anyunbound material and the bound test agent or bound MT-SP1 protein ornucleic acid is detected (e.g. by detection of a label attached to thebound molecule). The amount of immobilized label is proportional to thedegree of binding between the MT-SP1 protein or nucleic acid and thetest agent.

C) High Throughput Screening for MT-SP1 Modulators (e.g., Therapeutics).

The assays for modulators of MT-SP1 expression and/or activity describedherein are also amenable to “high-throughput” modalities.Conventionally, new chemical entities with useful properties (e.g.,modulation of MT-SP1 activity or expression) are generated byidentifying a chemical compound (called a “lead compound”) with somedesirable property or activity, creating variants of the lead compound,and evaluating the property and activity of those variant compounds.However, the current trend is to shorten the time scale for all aspectsof drug discovery. Because of the ability to test large numbers quicklyand efficiently, high throughput screening (HTS) methods are replacingconventional lead compound identification methods.

In one preferred embodiment, high throughput screening methods involveproviding a library containing a large number of compounds (candidatecompounds) potentially having the desired activity. Such “combinatorialchemical libraries” are then screened in one or more assays, asdescribed herein, to identify those library members (particular chemicalspecies or subclasses) that display a desired characteristic activity.The compounds thus identified can serve as conventional “lead compounds”or can themselves be used as potential or actual therapeutics.

1) Combinatorial Chemical Libraries for Potential Modulators of MT-SP1.

The likelihood of an assay identifying a MT-SP1 expression or activitymodulator is increased when the number and types of test agents used inthe screening system is increased. Recently, attention has focused onthe use of combinatorial chemical libraries to assist in the generationof new chemical compound leads. A combinatorial chemical library is acollection of diverse chemical compounds generated by either chemicalsynthesis or biological synthesis by combining a number of chemical“building blocks” such as reagents. For example, a linear combinatorialchemical library such as a polypeptide library is formed by combining aset of chemical building blocks called amino acids in every possible wayfor a given compound length (i.e., the number of amino acids in apolypeptide compound). Millions of chemical compounds can be synthesizedthrough such combinatorial mixing of chemical building blocks. Forexample, one commentator has observed that the systematic, combinatorialmixing of 100 interchangeable chemical building blocks results in thetheoretical synthesis of 100 million tetrameric compounds or 10 billionpentameric compounds (Gallop et al. (1994) 37(9): 1233-1250).

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37:487-493, Houghton et al. (1991) Nature, 354: 84-88). Peptide synthesisis by no means the only approach envisioned and intended for use withthe present invention. Other chemistries for generating chemicaldiversity libraries can also be used. Such chemistries include, but arenot limited to: peptoids (PCT Publication No WO 91/19735, 26 Dec. 1991),encoded peptides (PCT Publication WO 93/20242, 14 Oct. 1993), randombio-oligomers (PCT Publication WO 92/00091, 9 Jan. 1992),benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993) Proc.Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides (Hagihara etal. (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimeticswith a Beta-D-Glucose scaffolding (Hirschmann et al., (1992) J. Amer.Chem. Soc. 114: 9217-9218), analogous organic syntheses of smallcompound libraries (Chen et al. (1994) J. Amer. Chem. Soc. 116: 2661),oligocarbamates (Cho, et al., (1993) Science 261:1303), and/or peptidylphosphonates (Campbell et al., (1994) J. Org. Chem. 59: 658). See,generally, Gordon et al., (1994) J. Med. Chem. 37:1385, nucleic acidlibraries (see, e.g., Strategene, Corp.), peptide nucleic acid libraries(see, e.g., U.S. Pat. No. 5,539,083) antibody libraries (see, e.g.,Vaughn et al. (1996) Nature Biotechnology, 14(3): 309-314), andPCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al. (1996)Science, 274: 1520-1522, and U.S. Pat. No. 5,593,853), and small organicmolecule libraries (see, e.g., benzodiazepines, Baum (1993) C&EN,January 18, page 33, isoprenoids U.S. Pat. No. 5,569,588,thiazolidinones and metathiazanones U.S. Pat. No. 5,549,974,pyrrolidines U.S. Pat. Nos. 5,525,735 and 5,519,134, morpholinocompounds U.S. Pat. No. 5,506,337, benzodiazepines 5,288,514, and thelike).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.).

A number of well known robotic systems have also been developed forsolution phase chemistries. These systems include automated workstationslike the automated synthesis apparatus developed by Takeda ChemicalIndustries, LTD. (Osaka, Japan) and many robotic systems utilizingrobotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca,Hewlett-Packard, Palo Alto, Calif.) which mimic the manual syntheticoperations performed by a chemist. Any of the above devices are suitablefor use with the present invention. The nature and implementation ofmodifications to these devices (if any) so that they can operate asdiscussed herein will be apparent to persons skilled in the relevantart. In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J., Asinex,Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

2) High Throughput Assays of Chemical Libraries for Modulators ofMT-SP1.

Any of the assays for agents that modulate MT-SP1 expression and/oractivity (e.g. that have potential therapeutic activity) are amenable tohigh throughput screening. As described above, having identified thenucleic acid whose expression is altered upon exposure to a drug ofabuse, likely modulators either inhibit expression of the gene product,or inhibit the activity of the expressed protein. Preferred assays thusdetect inhibition of transcription (i.e., inhibition of mRNA production)by the test compound(s), inhibition of protein expression by the testcompound(s), or binding to the gene (e.g., gDNA, or cDNA) or geneproduct (e.g., mRNA or expressed protein) by the test compound(s).Alternatively, the assay can detect inhibition of the characteristicprotease activity of the MT-SP1 gene product. High throughput assays forthe presence, absence, or quantification of particular nucleic acids orprotein products are well known to those of skill in the art. Similarly,binding assays are similarly well known. Thus, for example, U.S. Pat.No. 5,559,410 discloses high throughput screening methods for proteins,U.S. Pat. No. 5,585,639 discloses high throughput screening methods fornucleic acid binding (i.e., in arrays), while U.S. Pat. Nos. 5,576,220and 5,541,061 disclose high throughput methods of screening forligand/antibody binding.

In addition, high throughput screening systems are commerciallyavailable (see, e.g., Zymark Corp., Hopkinton, Mass.; Air TechnicalIndustries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.;Precision Systems, Inc., Natick, Me., etc.). These systems typicallyautomate entire procedures including all sample and reagent pipetting,liquid dispensing, timed incubations, and final readings of themicroplate in detector(s) appropriate for the assay. These configurablesystems provide high throughput and rapid start up as well as a highdegree of flexibility and customization. The manufacturers of suchsystems provide detailed protocols the various high throughput. Thus,for example, Zymark Corp. provides technical bulletins describingscreening systems for detecting the modulation of gene transcription,ligand binding, and the like.

VI. Assay Optimization.

The assays of this invention have immediate utility in detectingelevated expression and/or activity of an MT-SP1 protease or forscreening for agents that modulate the MT-SP1 activity of a cell, tissueor organism. The assays of this invention can be optimized for use inparticular contexts, depending, for example, on the source and/or natureof the biological sample and/or the particular test agents, and/or theanalytic facilities available.

Thus, for example, optimization can involve determining optimalconditions for binding assays, optimum sample processing conditions(e.g. preferred PCR conditions), hybridization conditions that maximizesignal to noise, protocols that improve throughput, etc. In addition,assay formats can be selected and/or optimized according to theavailability of equipment and/or reagents. Thus, for example, wherecommercial antibodies or ELISA kits are available it may be desired toassay protein concentration. Conversely, where it is desired to screenfor modulators that alter transcription of one or more of the genes orESTs identified herein, nucleic acid based assays are preferred.

Routine selection and optimization of assay formats is well known tothose of ordinary skill in the art.

VII. MT-SP1-Targeted Therapeutics.

Since MT-SP1 is found in a cell membrane, it can be exploited as targetfor the efficient and specific delivery of an effector (e.g. an effectormolecule such as a cytotoxin, a radiolabel, etc.) to a cell expressingMT-SP1. In one preferred embodiment, chimeric molecules are used todeliver the effector to the cancer cell (or proliferating endothelialcell participating in angiogeneisis).

In a chimeric molecule, two or more molecules that exist separately intheir native state are joined together to form a single molecule havingthe desired functionality of all of its constituent molecules.Typically, one of the constituent molecules of a chimeric molecule is a“targeting molecule”. The targeting molecule is a molecule such as aligand or an antibody that specifically binds to its correspondingtarget, in this case an MT-SP1 protein.

Another constituent of the chimeric molecule is an “effector”. Theeffector molecule refers to a molecule or group of molecules that is tobe specifically transported to the target cell (e.g., a cell expressingan MT-SP1 polypeptide). The effector molecule typically has acharacteristic activity that is desired to be delivered to the targetcell. Effector molecules include, but are not limited to cytotoxins,labels, radionuclides, ligands, antibodies, drugs, liposomes, and thelike.

In particular, where the effector component is a cytotoxin, the chimericmolecule may act as a potent cell-killing agent specifically targetingthe cytotoxin to cells bearing a particular target molecule. Forexample, chimeric fusion proteins which include interleukin 4 (IL-4) ortransforming growth factor (TGFα) fused to Pseudomonas exotoxin (PE) orinterleukin 2 (IL-2) fused to Diphtheria toxin (DT) have been shown tospecifically target and kill cancer cells (Pastan et al., Ann. Rev.Biochem., 61: 331-354 (1992)).

A) The Targeting Molecule.

In a preferred embodiment, in the methods and compositions of thisinvention, the targeting molecule is an antibody that specifically bindsto a MT-SP1 protein or to a fragment thereof. The antibody can be afull-length antibody polyclonal or monoclonal antibody, an antibodyfragment (e.g. Fv, Fab, etc.), or a single chain antibody (e.g. scFv).

The antibody can be produced according to standar methods well known tothose of skill in the art as described above. The antibody once producedcan be chemically conjugated to the effector.

Where one of the effector molecule(s) is a protein, the antibody can bea single chain antibody and the chimeric molecule can be a recombinantlyexpressed fusion protein. Means of producing such recombinant fusionproteins are well known to those of skill in the art.

B) The Effector Molecule.

As described above, the effector molecule component of the chimericmolecules of this invention may be any molecule whose activity it isdesired to deliver to cells that express or overexpress a MT-SP1protein. Particularly preferred effector molecules include cytotoxinssuch as Pseudomonas exotoxin, or Diphtheria toxin, radionuclides,radio-sensitizing agents, ligands such as growth factors, antibodies,detectable labels such as fluorescent, radio-opaque, or radioactivelabels, and therapeutic compositions such as liposomes and variousdrugs.

1) Cytotoxins.

Particularly preferred cytotoxins include Pseudomonas exotokins,Diphtheria toxins, ricin, and abrin. Pseudomonas exotoxin and Dipthteriatoxin are most preferred.

Pseudomonas exotoxin A (PE) is an extremely active monomeric protein(molecular weight 66 kD), secreted by Pseudomonas aeruginosa, whichinhibits protein synthesis in eukaryotic cells through the inactivationof elongation factor 2 (EF-2) by catalyzing its ADP-ribosylation(catalyzing the transfer of the ADP ribosyl moiety of oxidized NAD ontoEF-2).

The toxin contains three structural domains that act in concert to causecytotoxicity. Domain Ia (amino acids 1-252) mediates cell binding.Domain II (amino acids 253-364) is responsible for translocation intothe cytosol and domain III (amino acids 400-613) mediates ADPribosylation of elongation factor 2, which inactivates the protein andcauses cell death. The function of domain Ib (amino acids 365-399)remains undefined, although a large part of it, amino acids 365-380, canbe deleted without loss of cytotoxicity. See Siegall et al., J. Biol.Chem. 264: 14256-14261 (1989).

Where the targeting molecule (e.g. anti-MT-SP1) is fused to PE, apreferred PE molecule is one in which domain Ia (amino acids 1 through252) is deleted and amino acids 365 to 380 have been deleted from domainIb. However all of domain Ib and a portion of domain II (amino acids 350to 394) can be deleted, particularly if the deleted sequences arereplaced with a linking peptide such as GGGGS (SEQ ID NO:77).

In addition, the PE molecules can be further modified usingsite-directed mutagenesis or other techniques known in the art, to alterthe molecule for a particular desired application. Means to alter the PEmolecule in a manner that does not substantially affect the functionaladvantages provided by the PE molecules described here can also be usedand such resulting molecules are intended to be covered herein.

For maximum cytotoxic properties of a preferred PE molecule, severalmodifications to the molecule are recommended. An appropriate carboxylterminal sequence to the recombinant molecule is preferred totranslocate the molecule into the cytosol of target cells. Amino acidsequences which have been found to be effective include, REDLK (SEQ IDNO:78) (as in native PE), REDL (SEQ ID NO:79) RDEL (SEQ ID NO:80), orKDEL (SEQ ID NO:81), repeats of those, or other sequences that functionto maintain or recycle proteins into the endoplasmic reticulum, referredto here as “endoplasmic retention sequences”. See, for example,Chaudhary et al. (1991) Proc. Natl. Acad. Sci. USA 87:308-312 andSeetharam et al, J. Biol. Chem. 266: 17376-17381. Preferred forms of PEcomprise the PE molecule designated PE38QQR. (Debinski et al. Bioconj.Chem., 5: 40 (1994)), and PE4E (see, e.g., Chaudhary et al. (1995) J.Biol. Chem., 265: 16306). The targeting molecule (e.g. anti-MT-SP1) mayalso be inserted at a point within domain III of the PE molecule or intodomain Ib. Methods of cloning genes encoding PE fused to various ligandsare well known to those of skill in the art (see, e.g., Siegall et al.,FASEB J, 3: 2647-2652 (1989); and Chaudhary et al. Proc. Natl. Acad.Sci. USA, 84: 4538-4542 (1987)).

Like PE, diphtheria toxin (DT) kills cells by ADP-ribosylatingelongation factor 2 thereby inhibiting protein synthesis. Diphtheriatoxin, however, is divided into two chains, A and B, linked by adisulfide bridge. In contrast to PE, chain B of DT, which is on thecarboxyl end, is responsible for receptor binding and chain A, which ispresent on the amino end, contains the enzymatic activity (Uchida etal., Science, 175: 901-903 (1972); Uchida et al. J. Biol. Chem., 248:3838-3844 (1973)).

In a preferred embodiment, the targeting molecule-Diphtheria toxinfusion proteins of this invention have the native receptor-bindingdomain removed by truncation of the Diphtheria toxin B chain.Particularly preferred is DT388, a DT in which the carboxyl terminalsequence beginning at residue 389 is removed. Chaudhary, et al., Bioch.Biophys. Res. Comm., 180: 545-551 (1991). Like the PE chimericcytotoxins, the DT molecules may be chemically conjugated to the MT-SP1antibody, but, in a preferred embodiment, the targeting molecule will befused to the Diphtheria toxin by recombinant means (see, e.g., Williamset al. (1990) J. Biol. Chem. 265: 11885-11889).

2) Detectable Labels.

Detectable labels suitable for use as the effector molecule component ofthe chimeric molecules of this invention include any compositiondetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical or chemical means. Useful labels in the presentinvention include magnetic beads (e.g. Dynabeads™), fluorescent dyes(e.g., fluorescein isothiocyanate, texas red, rhodamine, greenfluorescent protein, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S,¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkalinephosphatase and others commonly used in an ELISA), and colorimetriclabels such as colloidal gold or colored glass or plastic (e.g.polystyrene, polypropylene, latex, etc.) beads.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

3) Ligands.

The effector molecule may also be a ligand or an antibody. Particularlypreferred ligand and antibodies are those that bind to surface markerson immune cells. Chimeric molecules utilizing such antibodies aseffector molecules act as bifunctional linkers establishing anassociation between the immune cells bearing binding partner for theligand or antibody and the tumor cells expressing the MT-SP1 protein.Suitable antibodies and growth factors are known to those of skill inthe art and include, but are not limited to, IL-2, IL-4, IL-6, IL-7,tumor necrosis factor (TNF), anti-Tac, TGFα, and the like.

4) Other Therapeutic Moieties.

Other suitable effector molecules include pharmacological agents orencapsulation systems containing various pharmacological agents. Thus,the targeting molecule of the chimeric molecule may be attached directlyto a drug that is to be delivered directly to the tumor. Such drugs arewell known to those of skill in the art and include, but are not limitedto, doxirubicin, vinblastine, genistein, an antisense molecule, and thelike.

Alternatively, the effector molecule may be an encapsulation system,such as a viral capsid, a liposome, or micelle that contains atherapeutic composition such as a drug, a nucleic acid (e.g. anantisense nucleic acid), or another therapeutic moiety that ispreferably shielded from direct exposure to the circulatory system.Means of preparing liposomes attached to antibodies are well known tothose of skill in the art. See, for example, U.S. Pat. No. 4,957,735,Connor et al., Pharm. Ther., 28: 341-365 (1985)

C) Attachment of the Targeting Molecule to the Effector Molecule.

One of skill will appreciate that the MT-SP1 targeting molecule andeffector molecules may be joined together in any order. Thus, where thetargeting molecule is a polypeptide, the effector molecule may be joinedto either the amino or carboxy termini of the targeting molecule. Thetargeting molecule may also be joined to an internal region of theeffector molecule, or conversely, the effector molecule may be joined toan internal location of the targeting molecule, as long as theattachment does not interfere with the respective activities of themolecules.

The targeting molecule and the effector molecule may be attached by anyof a number of means well known to those of skill in the art. Typicallythe effector molecule is conjugated, either directly or through a linker(spacer), to the targeting molecule. However, where both the effectormolecule and the targeting molecule are polypeptides it is preferable torecombinantly express the chimeric molecule as a single-chain fusionprotein.

1) Conjugation of the Effector Molecule to the Targeting Molecule.

In one embodiment, the targeting molecule (e.g., anti-MT-SP1Ab) ischemically conjugated to the effector molecule (e.g., a cytotoxin, alabel, a ligand, or a drug or liposome). Means of chemically conjugatingmolecules are well known to those of skill.

The procedure for attaching an agent to an antibody or other polypeptidetargeting molecule will vary according to the chemical structure of theagent. Polypeptides typically contain variety of functional groups;e.g., carboxylic acid (COOH) or free amine (—NH₂) groups, which areavailable for reaction with a suitable functional group on an effectormolecule to bind the effector thereto.

Alternatively, the targeting molecule and/or effector molecule may bederivatized to expose or attach additional reactive functional groups.The derivatization may involve attachment of any of a number of linkermolecules such as those available from Pierce Chemical Company, RockfordIll.

A “linker”, as used herein, is a molecule that is used to join thetargeting molecule to the effector molecule. The linker is capable offorming covalent bonds to both the targeting molecule and to theeffector molecule. Suitable linkers are well known to those of skill inthe art and include, but are not limited to, straight or branched-chaincarbon linkers, heterocyclic carbon linkers, or peptide linkers. Wherethe targeting molecule and the effector molecule are polypeptides, thelinkers may be joined to the constituent amino acids through their sidegroups (e.g., through a disulfide linkage to cysteine). However, in apreferred embodiment, the linkers will be joined to the alpha carbonamino and carboxyl groups of the terminal amino acids.

A bifunctional linker having one functional group reactive with a groupon a particular agent, and another group reactive with an antibody, maybe used to form the desired immunoconjugate. Alternatively,derivatization may involve chemical treatment of the targeting molecule,e.g., glycol cleavage of the sugar moiety of a the glycoprotein antibodywith periodate to generate free aldehyde groups. The free aldehydegroups on the antibody may be reacted with free amine or hydrazinegroups on an agent to bind the agent thereto. (See U.S. Pat. No.4,671,958). Procedures for generation of free sulfhydryl groups onpolypeptide, such as antibodies or antibody fragments, are also known(See U.S. Pat. No. 4,659,839).

Many procedure and linker molecules for attachment of various compoundsincluding radionuclide metal chelates, toxins and drugs to proteins suchas antibodies are known (see, e.g., European Patent Application No.188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784;4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al. (1987)Cancer Res. 47: 4071-4075). In particular, production of variousimmunotoxins is well-known within the art and can be found, for examplein “Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet,”Thorpe et al., Monoclonal Antibodies in Clinical Medicine, AcademicPress, pp. 168-190 (1982), Waldmann (1991) Science, 252: 1657, U.S. Pat.Nos. 4,545,985 and 4,894,443.

In some circumstances, it is desirable to free the effector moleculefrom the targeting molecule when the chimeric molecule has reached itstarget site. Therefore, chimeric conjugates comprising linkages whichare cleavable in the vicinity of the target site may be used when theeffector is to be released at the target site. Cleaving of the linkageto release the agent from the antibody may be prompted by enzymaticactivity or conditions to which the immunoconjugate is subjected eitherinside the target cell or in the vicinity of the target site. When thetarget site is a tumor, a linker which is cleavable under conditionspresent at the tumor site (e.g. when exposed to tumor-associated enzymesor acidic pH) may be used.

A number of different cleavable linkers are known to those of skill inthe art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. Themechanisms for release of an agent from these linker groups include, forexample, irradiation of a photolabile bond and acid-catalyzedhydrolysis. U.S. Pat. No. 4,671,958, for example, includes a descriptionof immunoconjugates comprising linkers which are cleaved at the targetsite in vivo by the proteolytic enzymes of the patient=s complementsystem. In view of the large number of methods that have been reportedfor attaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, drugs, toxins, and other agents to antibodies one skilled inthe art will be able to determine a suitable method for attaching agiven agent to an antibody or other polypeptide.

2) Production of Fusion Proteins.

Where the MT-SP1 targeting molecule and/or the effector molecule isrelatively short (i.e., less than about 50 amino acids) they may besynthesized using standard chemical peptide synthesis techniques. Whereboth molecules are relatively short the chimeric molecule may besynthesized as a single contiguous polypeptide. Alternatively thetargeting molecule and the effector molecule may be synthesizedseparately and then fused by condensation of the amino terminus of onemolecule with the carboxyl terminus of the other molecule therebyforming a peptide bond. Alternatively, the targeting and effectormolecules may each be condensed with one end of a peptide spacermolecule thereby forming a contiguous fusion protein.

Solid phase synthesis in which the C-terminal amino acid of the sequenceis attached to an insoluble support followed by sequential addition ofthe remaining amino acids in the sequence is the preferred method forthe chemical synthesis of the polypeptides of this invention. Techniquesfor solid phase synthesis are described by Barany and Merrifield,Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963), andStewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co.,Rockford, Ill. (1984).

In a preferred embodiment, the chimeric fusion proteins of the presentinvention are synthesized using recombinant DNA methodology. Generallythis involves creating a DNA sequence that encodes the fusion protein,placing the DNA in an expression cassette under the control of aparticular promoter, expressing the protein in a host, isolating theexpressed protein and, if required, renaturing the protein.

DNA encoding the fusion proteins (e.g. anti-MT-SP1-PE38QQR) of thisinvention may be prepared by any suitable method, including, forexample, cloning and restriction of appropriate sequences or directchemical synthesis by methods such as the phosphotriester method ofNarang et al. Meth. Enzymol. 68: 90-99 (1979); the phosphodiester methodof Brown et al., Meth. Enzymol. 68: 109-151 (1979); thediethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859-1862 (1981); and the solid support method of U.S. Pat. No.4,458,066.

Chemical synthesis produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Alternatively, subsequences may be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments may then be ligated to produce the desired DNA sequence.

n a preferred embodiment, DNA encoding fusion proteins of the presentinvention may be cloned using DNA amplification methods such aspolymerase chain reaction (PCR). Thus, for example, the nucleic acidencoding an anti-MT-SP1 is PCR amplified, using a sense primercontaining the restriction site for NdeI and an antisense primercontaining the restriction site for HindIII. This produces a nucleicacid encoding the anti-MT-SP1 sequence and having terminal restrictionsites. A PE38QQR fragment may be cut out of the plasmid pWDMH4-38QQR orplasmid pSGC242FdN1 described by Debinski et al. (1994) Int. J. Cancer,58: 744-748. Ligation of the anti-MT-SP1 and PE38QQR sequences andinsertion into a vector produces a vector encoding anti-MT-SP1 joined tothe amino terminus of PE38QQR (position 253 of PE). The two moleculesare joined by a three amino acid junction consisting of glutamic acid,alanine, and phenylalanine introduced by the restriction site.

While the two molecules are preferably essentially directly joinedtogether, one of skill will appreciate that the molecules may beseparated by a peptide spacer consisting of one or more amino acids.Generally the spacer will have no specific biological activity otherthan to join the proteins or to preserve some minimum distance or otherspatial relationship between them. However, the constituent amino acidsof the spacer may be selected to influence some property of the moleculesuch as the folding, net charge, or hydrophobicity.

The nucleic acid sequences encoding the fusion proteins may be expressedin a variety of host cells, including E. coli, other bacterial hosts,yeast, and various higher eukaryotic cells such as the COS, CHO and HeLacells lines and myeloma cell lines. The recombinant protein gene will beoperably linked to appropriate expression control sequences for eachhost. For E. coli this includes a promoter such as the T7, tip, orlambda promoters, a ribosome binding site and preferably a transcriptiontermination signal. For eukaryotic cells, the control sequences willinclude a promoter and preferably an enhancer derived fromimmunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylationsequence, and may include splice donor and acceptor sequences.

The plasmids of the invention can be transferred into the chosen hostcell by well-known methods such as calcium chloride transformation forE. coli and calcium phosphate treatment or electroporation for mammaliancells. Cells transformed by the plasmids can be selected by resistanceto antibiotics conferred by genes contained on the plasmids, such as theamp, gpt, neo and hyg genes.

Once expressed, the recombinant fusion proteins can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982), Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y. (1990)). Substantially pure compositions of at least about 90to 95% homogeneity are preferred, and 98 to 99% or more homogeneity aremost preferred for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, the polypeptides may then be usedtherapeutically.

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the MT-SP1 targeted fusionprotein may possess a conformation substantially different than thenative conformations of the constituent polypeptides. In this case, itmay be necessary to denature and reduce the polypeptide and then tocause the polypeptide to re-fold into the preferred conformation.Methods of reducing and denaturing proteins and inducing re-folding arewell known to those of skill in the art (See, Debinski et al. (1993) J.Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug.Chem., 4: 581-585; and Buchner, et al. (1992) Anal. Biochem., 205:263-270).

One of skill would recognize that modifications can be made to theMT-SP1 targeted fusion proteins without diminishing their biologicalactivity. Some modifications may be made to facilitate the cloning,expression, or incorporation of the targeting molecule into a fusionprotein. Such modifications are well known to those of skill in the artand include, for example, a methionine added at the amino terminus toprovide an initiation site, or additional amino acids placed on eitherterminus to create conveniently located restriction sites or terminationcodons.

D) Pharmaceutical Compositions.

The chimeric molecules of this invention are useful for parenteral,topical, oral, or local administration (e.g. injected into a tumorsite), aerosol administration, or transdermal administration, forprophylactic, but principly for therapeutic treatment. Thepharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. For example,unit dosage forms suitable for oral administration include powder,tablets, pills, capsules and lozenges. It is recognized that the fusionproteins and pharmaceutical compositions of this invention, whenadministered orally, must be protected from digestion. This is typicallyaccomplished either by complexing the protein with a composition torender it resistant to acidic and enzymatic hydrolysis or by packagingthe protein in an appropriately resistant carrier such as a liposome.Means of protecting proteins from digestion are well known in the art.

The pharmaceutical compositions of this invention are particularlyuseful for parenteral administration, such as intravenous administrationor administration into a body cavity or lumen of an organ. Thecompositions for administration will commonly comprise a solution of thechimeric molecule dissolved in a pharmaceutically acceptable carrier,preferably an aqueous carrier. A variety of aqueous carriers can beused, e.g., buffered saline and the like. These solutions are sterileand generally free of undesirable matter. These compositions may besterilized by conventional, well known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example, sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like. The concentration ofchimeric molecule in these formulations can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight andthe like in accordance with the particular mode of administrationselected and the patient's needs.

Thus, a typical pharmaceutical composition for intravenousadministration would be about 0.1 to 10 mg per patient per day. Dosagesfrom 0.1 up to about 100 mg per patient per day may be used,particularly when the drug is administered to a secluded site and notinto the blood stream, such as into a body cavity or into a lumen of anorgan. Actual methods for preparing parenterally administrablecompositions will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington'sPharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa.(1980).

The compositions containing the present fusion proteins or a cocktailthereof (i.e., with other proteins) can be administered for therapeutictreatments. In therapeutic applications, compositions are administeredto a patient suffering from a disease, e.g., a cancer, in an amountsufficient to cure or at least partially arrest the disease and itscomplications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health.

Single or multiple administrations of the compositions may beadministered depending on the dosage and frequency as required andtolerated by the patient. In any event, the composition should provide asufficient quantity of the proteins of this invention to effectivelytreat the patient.

It will be appreciated by one of skill in the art that there are someregions that are not heavily vascularized or that are protected by cellsjoined by tight junctions and/or active transport mechanisms whichreduce or prevent the entry of macromolecules present in the bloodstream. Thus, for example, systemic administration of therapeutics totreat gliomas, or other brain cancers, is constrained by the blood-brainbarrier which resists the entry of macromolecules into the subarachnoidspace.

One of skill in the art will appreciate that in these instances, thetherapeutic compositions of this invention can be administered directlyto the tumor site. Thus, for example, brain tumors (e.g., gliomas) canbe treated by administering the therapeutic composition directly to thetumor site (e.g., through a surgically implanted catheter). Where thefluid delivery through the catheter is pressurized, small molecules(e.g. the therapeutic molecules of this invention) will typicallyinfiltrate as much as two to three centimeters beyond the tumor margin.

Alternatively, the therapeutic composition can be placed at the targetsite in a slow release formulation. Such formulations can include, forexample, a biocompatible sponge or other inert or resorbable matrixmaterial impregnated with the therapeutic composition, slow dissolvingtime release capsules or microcapsules, and the like.

Typically the catheter or time release formulation will be placed at thetumor site as part of a surgical procedure. Thus, for example, wheremajor tumor mass is surgically removed, the perfusing catheter or timerelease formulation can be emplaced at the tumor site as an adjuncttherapy. Of course, surgical removal of the tumor mass may be undesired,not required, or impossible, in which case, the delivery of thetherapeutic compositions of this invention may comprise the primarytherapeutic modality.

E) Tumor Imaging and Radio-Sensitizing Compositions.

1) Imaging Compositions.

In certain embodiments, the chimeric molecules of this invention can beused to direct detectable labels to a tumor site. This can facilitatetumor detection and/or localization. In a particularly preferredembodiment, the effector compoent of the chimeric molecule isa“radiopaque” label, e.g. a label that can be easily visualized usingx-rays. Radiopaque materials are well known to those of skill in theart. The most common radiopaque materials include iodide, bromide orbarium salts. Other radiopaque materials are also known and include, butare not limited to organic bismuth derivatives (see, e.g., U.S. Pat. No.5,939,045), radiopaque polyurethanes (see U.S. Pat. No. 5,346,9810,organobismuth composites (see, e.g., U.S. Pat. No. 5,256,334),radiopaque barium polymer complexes (see, e.g., U.S. Pat. No.4,866,132), and the like.

The anti-MT-SP1 antibodie(s) can be coupled directly to the radiopaquemoiety or they can be attached to a “package” (e.g. a liposome, apolymer microbead, etc.) carrying or containing the radiopaque material.

2) Radiosensitizers.

In another embodiment, the effector can be a radiosensitizer thatenhances the cytotoxic effect of ionizing radiation (e.g., such as mightbe produced by ⁶⁰Co or an x-ray source) on a cell. Numerousradiosensitizing agents are known and include, but are not limited tobenzoporphyrin derivative compounds (see, e.g., U.S. Pat. No.5,945,439), 1,2,4-benzotriazine oxides (see, e.g., U.S. Pat. No.5,849,738), compounds containing certain diamines (see, e.g., U.S. Pat.No. 5,700,825), BCNT (see, e.g., U.S. Pat. No. 5,872,107),radiosensitizing nitrobenzoic acid amide derivatives (see, e.g., U.S.Pat. No. 4,474,814), various heterocyclic derivatives (see, e.g., U.S.Pat. No. 5,064,849), platinum complexes (see, e.g., U.S. Pat. No.4,921,963), and the like.

The anti-MT-SP1 antibodie(s) can be coupled directly to the radiopaquemoiety or they can be attached to a “package” (e.g. a liposome, apolymer microbead, etc.) carrying or containing the radiosensitizingmaterial.

VIII. Kits.

In still another embodiment, this invention provides kits for practiceof the assays or use of the therapeutics and/or diagnostics describedherein. In one preferred embodiment, the kits comprise one or morecontainers containing antibodies and/or nucleic acid probes and/orsubstrates suitable for detection of MT-SP1 proteins or proteinfragments, and/or MT-SP1 nucleic acid(s), and/or and MT-SP1 proteinactivity, respectively. In other embodiments, the kits include one ormore of the MT-SP1 directed chimeric molecules discussed herein. Thekits may optionally include any reagents and/or apparatus to facilitatepractice of the assays or delivery of the molecules described herein.Such reagents include, but are not limited to buffers, pharmacologicalexcipients, labels, labeled antibodies, labeled nucleic acids, filtersets for visualization of fluorescent labels, blotting membranes, andthe like.

In addition, the kits may include instructional materials containingdirections (i.e., protocols) for the practice of the assay methods oruse of the chimeric molecules of this invention. Preferred instructionalmaterials provide protocols for assaying MT-SP1 gene expression, and/orprotein levels, and/or MT-SP1 protein activity, while other preferredinstructional materials provide guidance and instructions for the use ofthe chimeric molecules described herein. While the instructionalmaterials typically comprise written or printed materials they are notlimited to such. Any medium capable of storing such instructions andcommunicating them to an end user is contemplated by this invention.Such media include, but are not limited to electronic storage media(e.g., magnetic discs, tapes, cartridges chips), optical media (e.g., CDROM), and the like. Such media may include addresses to internet sitesthat provide such instructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Reverse Biochemistry: Using Macromolecular Protease Inhibitorsto Identify a Membrane-Type Serine Protease in Epithelial Cancer andNormal Tissue

This example describes the use of a “fold-specific” inhibitor (^(i),^(ii)) in studying the role of these chymotrypsin-fold serine proteasesin cancer. Ecotin or engineered versions of ecotin are introduced intocomplex biological systems as probes of proteolysis by thesechymotrypsin-fold proteases. When, as demonstrated herein, effects areobserved upon treatment with these unique inhibitors, then the largebody of knowledge concerning the biochemistry of these proteases can betapped to understand the structure and function of the target proteases.

For example, the molecular cloning, structural modeling, and mechanisticunderstanding of the enzymes are immediately accessible. Analogous to“reverse genetics” we refer to this approach as “reverse biochemistry”and have applied it to identify specific serine proteases in prostatecancer.

One useful model system for studying many issues that are pertinent toprostate cancer is the development of the rodent ventral prostate (VP)in explant cultures. Macromolecular inhibitors of serine proteases ofthe chymotrypsin fold, ecotin and ecotin M84R/M85R (see copendingapplication Ser. Nos. 09/290,513 and 09/289,830, both filed on Apr. 12,1999), inhibit ductal branching morphogenesis and differentiation of theexplanted rat VP. Ecotin M84R/M85R is an 2800-fold more potent inhibitorof uPA compared to ecotin (1 nM and 2.8 μM respectively). However,inhibition of prostate differentiation was seen with both inhibitors,suggesting that uPA and other related serine proteases are involved inthe differentiation and continued growth of the rat VP. Thusunidentified serine proteases may play a role in growth and preventionof apoptosis in prostate epithelial cells in this system.

Another well characterized model that is derived from human prostatecancer epithelial cells is the PC-3 cell line (Kaighn et al. (1979)Invest. Urology 17: 16-23). The PC-3 cell line expresses uPA as assayedby enzyme-linked immunosorbent assay (ELISA) and by Northern blotting ofPC-3 mRNA (Yoshida et al. (1994) Cancer Res. 54: 3300-3304). We foundthat the primary tumor size in PC-3 implanted nude mice wassignificantly smaller in ecotin M84R/M85R and ecotin wild-type treatedmice treated for seven weeks compared to the primary tumor size ofPBS-treated mice after four weeks. Metastasis from the primary tumorssimilarly were similarly lower in the inhibitor-treated mice compared toPBS treated mice. Inhibition was not unexpected with ecotin M84R/M85Rtreatment, since uPA has been implicated in metastasis. However,wild-type ecotin is a poor, micromolar inhibitor of uPA; oneinterpretation of the data is that the decrease in tumor size andmetastasis in the mouse model involves the inhibition of additionalserine proteases. Thus identification of the serine proteases expressedby PC-3 prostate cells may provide insight into the role of theseproteases in cancer and prostate growth and development. In this examplewe have extended the strategy of using the polymerase chain reaction(PCR) with degenerate oligonucleotide primers that were designed usingconserved sequence homology (Sakanari et al. (1989) Proc. Natl. Acad.Sci. USA 86: 4863-4867; Wiegand et al. (1993) Gene 136: 167-175, Kang etal. (1992) Gene 110: 181-187) to identify additional serine proteasesmade by cancer cells. Five independent serine protease cDNAs derivedfrom PC-3 mRNA were sequenced, including a novel serine protease, whichwe refer to as membrane-type serine protease 1 (MT-SP1), and the cloningand characterization of this cDNA that encodes a mosaic, transmembraneprotease is reported.

Materials and Methods

Materials

All primers used were synthesized on a Applied Biosystems 391 DNAsynthesizer. All restriction enzymes were purchased from New EnglandBiolabs. Automated DNA sequencing was carried out on an AppliedBiosystems 377 Prism sequencer, and manual DNA sequencing was carriedout under standard conditions. N-terminal amino acid sequencing wasperformed on an ABI 477A by the Biomolecular resource center. Thesynthetic substrates, Suc-AAPX-pNA,[N-succinyl-alanyl-alanyl-prolyl-Xxx-pNA (Xxx=alanyl, aspartyl,glutamyl, phenylalanyl, leucinyl, methionyl, and arginyl)], andH-Arg-pNA, (arginyl-pNA), were purchased from Bachem. Deglycosylationwas performed using PNGase F (NEB). All other reagents were of thehighest quality available and purchased from Sigma or Fisher unlessotherwise noted.

Isolation of cDNA from PC-3 Cells

mRNA was isolated from PC-3 cells using the polyATtract System 1000 kit(Promega). Reverse transcription was primed using the “lock-docking”oligo dT primer (Borsont et al. (1992) PCR Meth. Appl. 2: 144-148).Superscript II reverse transcriptase (Life Technologies) was used inaccordance with the manufacturer's instructions to synthesize the cDNAfrom the PC-3 mRNA.

Amplification of MT-SP1 Gene

The degenerate primers used for amplifying the protease domains weredesigned from the consensus sequences flanking the catalytic histidine(5′ His-primer) and the catalytic serine (3′ Ser-primer), similar tothose described (Sakanari et al. (1989) Proc. Natl. Acad. Sci. USA 86,4863-4867). The 5′ primer used is as follows: 5′-TGG (AG)TI (CAG)TI(AT)(GC)I GCI (GA)CI CA(CT) TG-3′ (SEQ ID NO: 3), where nucleotides inparentheses represent equimolar mixtures, and I represents deoxyinosine.This primer encodes at least the following amino acid sequence: W (I/V)(I/V/L/M) (S/T) A (A/T) H C (SEQ ID NO: 4). The 3′ primer used is asfollows: 5′-IGG ICC ICC I(GC)(AT) (AG)TC ICC (CT)TI (GA)CA IG(ATC)(GA)TC-3′ (SEQ ID NO: 5). The reverse complement of the 3′ primerencodes at least the following amino acid sequence: D (A/S/T) C(K/E/Q/H) G D S G G P(SEQ ID NO:6).

Direct amplification of serine protease cDNA was not possible using theabove primers. Instead, the first PCR was performed with the5′-His-primer and the oligo dT primer described above, using the“touchdown” PCR protocol (Don et al. (1991) Nucleic Acids Res. 19:4008), with annealing temperatures decreasing from 52° C. to 42° C. over22 rounds, and 13 final rounds at 54° C. annealing temperature. Cycletimes were 1 minute denaturing, 1 minute annealing, and 2 minuteextension times, followed by one final extension time of 15 minutesafter the final round of PCR. The template for the second PCR was 0.5 μL(total reaction volume 50 μL) of a 1 to 16 dilution of the first PCRreaction mixture that was performed with the 5′ His-primer and the oligodT. The second PCR reaction was primed with the 5′ His and the 3′Ser-primers and performed using the touchdown protocol described above.All PCR reactions used 12.5 pmol of primer for 50 μL reaction volume.

The product of the second reaction was purified on a 2% agarose gel, andall products between 400 and 550 base pairs were cut from the gel andextracted using the Qiaquick gel extraction kit (Qiagen). These productswere digested with the BamHI restriction enzyme to cut any uPA cDNA, andall 400-500 bp fragments were repurified on a 2% agarose gel. Thesereaction products were subjected to a third PCR using the 5′ His-primerand the 3′ Ser-primer using the identical touchdown procedure. Thesereaction products were gel purified and directly cloned into the pPCR2.1vector using the TOPO TA ligation kit (Invitrogen). DNA sequencing ofthe inserts determined the cDNA sequence from nucleotides 1984-2460, seeFIG. 1.

Northern Blot Analysis

³²P-Labeled nucleotides were purchased from Amersham Life Sciences. AcDNA fragment containing nucleotides 1173-2510 was digested from ESTw39209 using restriction enzymes EcoRI and BsmbI, yielding a 1.3 kbnucleotide insert. Labeled cDNA probes were synthesized using theRediprime random primer labelling kit (Amersham) and 20 ng of thepurified insert. Poly(A)+RNA membranes for Northern blotting werepurchased from Origene (HB-1002, HB-1018) and Clontech (Human II#7759-1, Human Cancer Cell Line #7757). The blots were performed understringent annealing conditions as described in (Ausubel et al. (ed.).(1990) Current protocols in molecular biology. Wiley & Sons, New York,N.Y.).

Construction of Expression Vectors

The mature protease domain and a small portion of the pro domain(nucleotides 1822-2601) cDNA were amplified using PCR from EST w39209and ligated into the pQE30 vector (Qiagen). This construct is designedto overexpress the protease sequence from amino acids (aa) 596-855 withthe following fusion: Met-Arg-Gly-Ser-His₆- (SEQ ID NO:82) aa596-855.The His-tag fusion allows affinity purification using metal chelatechromatography. The change from Ser⁸⁰⁵, encoded by TCC, to Ala (GCT) wasperformed using PCR. The presence of the correct Ser to Ala substitutionin the pQE30 vector was verified by DNA sequence analysis.

Expression and Purification of the Protease Domain

The above-mentioned plasmids were separately transformed into E. coliX-90 to afford high-level expression of recombinant protease geneproducts (Evnin et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6659-6663).Expression and purification of the recombinant enzyme from solubilizedinclusion bodies was performed as described previously (Unal et al.(1997) J. Virol. 71, 7030-7038). Protein containing fractions werepooled and dialyzed overnight at 4° C. against 50 mM Tris pH 8, 10%glycerol, 1 mM □-mercaptoethanol, 3M urea. Autoactivation of theprotease was monitored upon dialysis against storage buffer (50 mM TrispH 8, 10% glycerol) at 4° C. using the substrate Spectrozyme tPA(hexahydrotyrosyl-Gly-Arg-pNA, American Diagnostica). Hydrolysis ofSpectrozyme tPA was monitored at 405 nM for the formation ofp-nitroaniline using a UVIKON 860 spectrophotometer. Activated proteasewas bound to an immobilized p-aminobenzamidine resin (Pierce) that hadbeen equilibrated with storage buffer. Bound protease was eluted with100 mM benzamidine and the protein containing fractions were pooled.Excess benzamidine was removed using FPLC with a Superdex 70 (Pharmacia)gel filtration column that was equilibrated with storage buffer. Proteincontaining fractions were pooled and stored at −80° C. The cleavage ofthe purified Ser⁸⁰⁵ Ala protease domain was performed at 37° C. byaddition of active recombinant protease domain to 10 nM. Cleavage wasmonitored by SDS-PAGE.

Determination of Substrate Kinetics

The purified serine protease domain was titrated with4-methylumbelliferyl p-guanidinobenzoate (MUGB) to obtain an accurateconcentration of enzyme active sites (^(iii)). Enzyme activity wasmonitored at 25° C. in assay buffer containing 50 mM Tris pH 8.8, 50 mMNaCl, and 0.01% Tween 20. The final concentration of substrateSpectrozyme tPA ranged from 1 μM-400 μM. Enzyme concentrations rangedfrom 40 pM-800 pM. Active site titrations were performed on aFluoromax-2 spectrofluorimeter. Measurements were plotted using theKaleidaGraph program (Synergy, Reading, Pa.), and the K_(m), k_(cat),and k_(cat)/K_(m) for Spectrozyme tPA was determined using theMichaelis-Menten equation.

Inhibition of MT-SP1 Protease Domain with Ecotin and Ecotin M84R/M85R

Ecotin and ecotin M84R/M85R were purified from E. coli as described incopending application Ser. Nos. 09/290,513 and 09/289,830, both filed onApr. 12, 1999. Various concentrations of ecotin or ecotin M84R/M85R wereincubated with the His-tagged serine protease domain in a total volumeof 990 μL of buffer containing 50 mM NaCl, 50 mM Tris-HCl (pH 8.8),0.01% Tween 20. 10 μL of Spectrozyme tPA was added, yielding a solutioncontaining 100 μM substrate. The final enzyme concentration was 63 pM,and the ecotin and ecotin M84R/M85R concentration ranged from 0.1 nM to50 nM. The data were fit to the equation derived for kinetics ofreversible tight-binding inhibitors (Morrison (1969) Biochim. Biophys.Acta 185: 269-286, Williams and Morrison (1979) Methods. Enzymol. 63:437-467), and the values for apparent K_(i) were determined.

Results

Cloning of Serine Protease Domain cDNAs from PC-3 Cells andAmplification of MT-SP1 cDNA

PCR amplification of serine protease cDNA was performed using “consensuscloning”, where the amplification was performed with degenerate primersdesigned to anneal to cDNA encoding the region about the conservedcatalytic histidine (5′ His-primer) and the conserved catalytic serine(3′ Ser-primer). The consensus primers were designed using 37 humansequences within a sequence aligment of 242 serine proteases of thechymotrypsin fold that are reported in the Swiss protein database. Inorder to bias the screen for previously unidentified proteases in thePC-3 cDNA, uPA cDNA was cut and removed using the known BamHIendonuclease site in the uPA cDNA sequence. The expected size of thecDNA fragments amplified between His⁵⁷ and Ser¹⁹⁵ cDNA (standardchymotrypsinogen numbering) is between 400-550 base pairs;statistically, only one in ten cDNAs of that length will be cleaved byBamHI. Thus, cDNAs obtained from the PCR reactions with the 5′His-primer and 3′ Ser-primer were size selected for the 400-550 bprange, digested with BamHI and purified from any digested cDNAs. After asubsequent round of PCR, the products were cloned into pPCR2.1 (FIG. 2).Twenty clones were digested with EcoRI to monitor the size of the cDNAinsert. Three clones lacked inserts of the correct size. The remainingseventeen clones containing inserts between 400 and 550 bp weresequenced. Blast searches of the resulting sequences revealed that sixclones did not match serine protease sequences. The remaining cDNAsyielded clones corresponding to factor XII (2 clones), protein C (2clones), trypsinogen type IV (2 clones), uPA (1 clone) and a cDNAdenoted as membrane-type serine protease 1 (MT-SP1) (4 clones).Additional serine protease sequences may not have been found becausethey were digested by BamHI, lost in the size selection, or were presentin lower frequencies.

Multiple EST sequences were found for the cDNA. EST accessions aa459076,aa219372, and w39209 were used extensively for sequencing the cDNAstarting from nucleotide 746, and 2461-3142, but no start codon wasobserved. A sequence was also found in GenBank, accession no. U20428.This sequence also lacks the 5′ end of the cDNA, but allowedamplification of cDNA from nucleotides 196-745. Rapid amplification ofcDNA ends (RACE) techniques (Frohman (1993) Methods Enzymol. 218:340-356) were used to obtain further 5′ cDNA sequence. Application ofRACE did not yield a clone containing the entire 5′ untranslated region,but the sequence obtained contained a stop codon in frame with the Kozakstart sequence (Kozak (1991) J. Cell Biol. 115: 887-903), givingconfidence that the full coding sequence of the cDNA has been obtained.The nucleotide sequence and predicted amino acid sequence are shown inFIG. 1 (SEQ ID NO: 1 and 2).

The nucleotide sequence surrounding the proposed start codon matches theoptimal sequence of ACCATGG (SEQ ID NO: 7) for translation initiationsites proposed by Kozak (supra.). In addition, there is a stop codon inframe with the putative start codon, which gives further evidence thatinitiation occurs at that site. The DNA sequence predicts an 855 aminoacid mosaic protein composed of multiple domains (FIG. 3). The codingsequence does not contain a typical signal peptide, but does contain asingle hydrophobic sequence of 26 residues (residues 55-81), which isflanked by a charged residue on each side. This sequence may constitutea signal anchor (SA) sequence, similar to that observed in otherproteases, including hepsin (Leytus et al. (1988) Biochemistry 27:1067-1074) and enteropeptidase (Kitamoto et al. (1994) Proc. Natl. Acad.Sci. USA 91: 7588-7592). Following the putative SA sequence are two CUBdomains (Bork and Beckmann (1993) J. Mol. Biol. 231: 539-545), which arenamed after the proteins in which the modules were first discovered:complement subcomponents C1s and C1r, urchin embryonic growth factor(Uegf), and bone morphogenetic protein 1 (EMP 1). CUB domains haveconserved characteristics, which include the presence of four cysteineresidues and various conserved hydrophobic and aromatic positions (Borkand Beckmann (1993) J. Mol. Biol. 231: 539-545). The CUB domain, whichhas recently been characterized crystallographically (Varela et al.(1997) J. Mol. Biol. 274: 635-649), consists of ten □-strands that areorganized into two 5-stranded β-sheets. Following the CUB domains arefour LDLR repeats (Krieger and Herz (1994) Annu. Rev. Biochem. 63:601-637), which are named after the receptor ligand-binding repeats thatare present in the LDL receptor. These repeats have a highly conservedpattern and spacing of six cysteine residues that form threeintramolecular disulfide bonds. The final domain observed is the serineprotease domain. The alignments of these domains with other members oftheir respective classes are shown in FIG. 4.

Tissue Distribution of MT-SP1 mRNA.

Northern blots of human poly(A)+ RNA, using a 1.3 kB fragment of MT-SP1cDNA fragment as a probe, show a ˜3.3 kB fragment appearing inepithelial tissues including the prostate, kidney, spleen, liver,leukocytes, lung, small intestine, stomach, thymus, colon, and placenta,and explants of human breast cancer and mastases. This band was notobserved in muscle, brain, ovary, or testis (FIG. 5). Similarexperiments performed on a human cancer cell line blot shows that MT-SP1is expressed in the Colorectal adenocarcinoma, SW480, and human breastcancer, but was not observed in the Promyelocytic Leukemia HL-60, HeLaCell S3, Chronic Myelogenous Leukemia K-562, Lymphoblastic LeukemiaMOLT-4, Burkitt's Lymphoma Raji, Lung Carcinoma A549, or Melanoma G361lanes (data not shown). MT-SP1 is also expressed in blood vessels ofprostatic and gastric cancers. This 3.3 kB mRNA fragment is slightlylonger than the 3.1 kB sequence presented in FIG. 5, suggesting thatthere may still be further sequence in the 5′ untranslated region thathas not been identified.

Activation and Purification of His-MT-SP1 Protease Domain

The serine protease domain of MT-SP1 was expressed in E. coli as aHis-tagged fusion, and was purified from inclusion bodies underdenaturing conditions using metal-chelate affinity chromatography. Theyield of enzyme after this step was approximately 3 mg of protein perliter of E. coli culture. This denatured protein refolded when the ureawas slowly dialyzed away from the protein. Surprisingly, the purifiedrenatured protein showed a time dependent shift on an SDS-PAGE gel (FIG.6A, lanes (a) 1-7), with the lower fragment being the size of themature, processed enzyme, lacking the His tag. N-terminal sequencing ofthe purified, activated protease domain yielded the expected VVGGT (SEQID NO:83) activation sequence. When the refolded protein was tested foractivity using the synthetic substratehexahydrotyrosyl-glycyl-arginyl-paranitroanilide (Spectrozyme tPA), atime dependent increase in activity was observed (FIG. 6B). In contrast,the protease domain that contains the Ser⁸⁰⁵ Ala mutation did not eithershow a change in size on an SDS polyacrylamide gel or an increase inenzymatic activity under identical conditions (data not shown),suggesting that the catalytic serine is necessary for activation and notthe result of a contaminating protease. In order to show that thecleavage of the protease domain was a result of His-tagged MT-SP1protease activity, the inactive Ser⁸⁰⁵ Ala protease domain was treatedwith purified recombinant enzyme (FIG. 6C). This treatment results inthe formation of a cleavage product that corresponds to the size of theactive protease (FIG. 6C, lane 7). Untreated protease domain does notget cleaved (FIG. 6C, lane 8). From these results, it is concluded thatthe protease autoactivates upon refolding. The activated protease wasseparated from inactive protein and other contaminants using affinitychromatography with p-aminobenzamidine resin. Purified protein wasanalyzed by SDS-PAGE and no other contaminants were observed. Similarly,immunoblotting with polyclonal antiserum against purified proteasedomain (raised in rabbits at Berkeley Antibody Company) revealed oneband. Under non-reducing conditions, the pro-region is disulfide linkedto the protease domain; thus, this purified protein was alsoimmunoreactive with the monoclonal antibody (Qiagen) directed againstthe amino-terminal Arg-Gly-Ser-His₄ (SEQ ID NO:34) epitope that iscontained in the recombinant protease domain, further indicating thepurity and identity of the protein (data not shown).

Kinetic Properties of Purified His-MT-SP1 Protease Domain

The enzyme concentration was determined using an active site titrationwith MUGB. The catalytic activity of the protease domain was monitoredusing pNA substrates. Purified protease domain was tested for hydrolyticactivity against tetrapeptide substrates of the form Suc-AAPX-pNA, whichcontained various amino acids at the P1 position (P1-Ala, Asp, Glu, Phe,Leu, Met, Lys, or Arg). The only substrates with detectable activitywere those with P1-Lys or P1-Arg. The serine protease domain with theSer⁸⁰⁵ Ala mutation had no detectable activity. The activity of theprotease domain was further characterized using the substrateSpectrozyme tPA (hexahydrotyrosyl-Gly-Arg-pNA), yielding: K_(m)=31.4±4.2μM, k_(cat)=2.6×10²±6.5 s⁻¹, and k_(cat)/K_(m)=6.9×10⁶±2.3×10⁶ M⁻¹ s⁻¹.Ecotin inhibition of the MT-SP1 His-tagged protease domain fits atight-binding reversible inhibitory model as observed for ecotininteraction with other serine protease targets. Inhibition assays usingecotin and ecotin M84R/M85R yielded apparent K_(i)'s of 782±92 pM and9.8±1.5 pM respectively.

Discussion

Structural Motifs of MT-SP1

In this work, we characterize the expression of chymotrypsin-foldproteases by PC-3 cells and cloned a member of this family we callMT-SP1. The name membrane-type serine protease 1 (MT-SP1) is given to beconsistent with the nomenclature of the membrane-type matrixmetalloproteases (MT-MMPs) (Nagase (1997) Biol. Chem. 378, 151-160). ThecDNA likely encodes a membrane-type protein due to the lack of a signalsequence and the presence of a putative signal anchor (SA) that is alsoseen in other membrane-type serine proteases hepsin (Leytus et al.(1988) Biochemistry 27: 1067-1074), enteropeptidase (Kitamoto et al.(1994) Proc. Natl. Acad. Sci. USA 91: 7588-7592), TMPRSS2(Poloni-Giacobino et al. (1997) Genomics 44, 309-320), and human airwaytrypsin-like protease (Yamakoka et al. (1998) J. Biol. Chem. 273,11895-11901). We propose that proteins that are localized to themembrane through a signal anchor and that encode a chymotrypsin foldserine protease domain be categorized in the MT-SP family. The membranelocalization of MT-SP1 is supported by immunofluorescence experimentsthat localize the protease domain to the extracellular cell surface.

Following the putative SA are several domains that are thought to beinvolved in protein-protein interactions or protein ligand interactions.For example, CUB domains can mediate protein-protein interactions, aswith the seminal plasma PSP-I/PSP-II heterodimer that is built by CUBdomain interactions and with procollagen C-proteinase enhancer proteinand procollagen C-proteinase (BMP-1) (Kessler and Adar (1989) Eur. J.Biochem. 186, 115-121; Hulmes et al. (1997) Matrix Biol. 16, 41-45).Interestingly, most of the proteins that contain CUB domains areinvolved in developmental processes or are involved in proteolyticcascades, which suggests that MT-SP1 may play a similar role. The fourrepeated motifs that follow the CUB domains are known as LDL receptorligand-binding repeats, named after the seven copies of repeats found inthe LDL receptor. There are several negatively charged amino acidsbetween the fourth and sixth cysteines that are highly conserved in theLDL receptor and also seen in the LDLR repeats of MT-SP1. The conservedmotif Ser-Asp-Glu (residues 44-46 in FIG. 4) are known to be importantfor binding the positively charged residues of the LDL receptor ligandsapolipoprotein B-100 (ApoB-100) and ApoE. The ligand binding repeats ofMT-SP1 most likely do not mediate interaction with ApoB-100 or ApoE, butmay be involved in the interaction with other positively chargedligands. For example, LDLR repeats in the LDL receptor-related proteinhave been implicated the binding and recycling of protease/inhibitorcomplexes such as uPA/plasminogen activator inhibitor-1 (PAI-1)complexes (reviewed in Strickl et al. (1995) FASEB J. 9, 890-898;Moestrup (1994) Biochim. Biopys. Acta 1197, 197-213). It also has beenshown that the pro domain of enteropeptidase is involved in interactionswith its substrate trypsinogen, allowing 520-fold greater catalyticefficiency in the cleavage compared to the protease domain alone (Lu etal. (1997) J. Biol. Chem. 272, 31293-31300). By analogy, similarinteractions should occur between MT-SP1 and its substrates. Thus,further investigation of MT-SP1 CUB domain or LDLR repeat interactionsmay yield insight into the function of this protein.

The amino acid sequence of the serine protease domain of MT-SP1 ishighly homologous to other proteases found in the family (FIG. 4). Theessential features of a functional serine protease are contained in thededuced amino acid sequence of the domain. The residues that comprisethe catalytic triad, His⁶⁵⁶, Asp⁷¹¹, Ser⁸⁰⁵, corresponding to His⁵⁷,Asp¹⁰², and Ser¹⁹⁵ in chymotrypsin, are observed in MT-SP1(see Peronaand Craik (1995) Protein Sci. 4: 337-360, Perona and Craik (1997) J.Biol. C hem. 272: 29987-29990 for reviews). The sequenceSer²¹⁴-Trp²¹⁵-Gly²¹⁶ (Ser⁸²⁵-Tr⁸²⁶-Gly⁸²⁷), which is thought to interactwith the side chains of the substrate for properly orienting thescissile bond is present. Gly¹⁹³ (Gly⁸⁰³) and Gly¹⁹⁶ (Gly⁸⁰⁵), which arethought to be necessary for proper orientation of Ser¹⁹⁵ (Ser⁸⁰⁵) alsoare present. Based upon homology to chymotrypsin, three disulfide bondsare predicted to form within the protease domain at Cys⁴⁴-Cys⁵⁸,Cys¹⁶⁸-Cys¹⁸², and Cys¹⁹¹-Cys²²⁰ (Cys⁶⁴³-Cys⁶⁵⁷, Cys⁷⁷⁶-Cys⁷⁹⁰,Cys⁸⁰¹-Cys⁸³⁰), and a fourth disulfide bond should form between thecatalytic and the pro-domain Cys¹²²-Cys¹ (Cys⁷³¹-Cys⁶⁰⁴), as observedfor chymotrypsin. This predicted disulfide with the pro-domain suggeststhat the active catalytic domain should still be localized to the cellsurface via a disulfide linkage. The presence of the catalytic machineryand other conserved structural components described above suggest thatall features necessary for proteolytic activity are present in theencoded sequence.

Substrate Specificity of the MT-SP1 Protease Domain

The S1 site specificity (Schecter and Berger (1967) Biochem. Biophys.Res. Commun. 27: 157-162) of a protease is largely determined by theamino acid residue at position 189. This position is occupied by anaspartate in MT-SP1, suggesting that the protease has specificity forArg/Lys in the P1 position. In addition, the presence of a polar Gln¹⁹²(Gln⁸⁰³), as in trypsin is consistent with basic specificity.Furthermore, the presence of Gly²¹⁶ (Gly⁸²⁷) and Gly²²⁶ (Gly⁸³⁷) isconsistent with the presence of a deep S1 pocket, unlike elastase, whichhas Val²¹⁶ and Thr²²⁶ that block the pocket and thereby contribute tothe P1 specificity for small hydrophobic side chains. The specificity atthe other subsites is largely dependent upon the nature of the sevenloops A-E and loops 2 and 3 (FIG. 4). Loop C in enterokinase has anumber of positively charged residues that are thought to interact withthe negatively-charged activation site in trypsinogen,Asp-Asp-Asp-Asp-Lys (SEQ ID NO:8). One known substrate for MT-SP1 (asdescribed below) is the activation site of MT-SP1, which isArg-Gln-Ala-Arg (residues 611-614). Loop C contains two aspartateresidues that may participate in the recognition of the activationsequence.

One means of obtaining further data on substrate specificity is bycharacterization of the activity of the recombinant proteolytic domain.Enterokinase has been characterized from both recombinant (LaVallie etal. (1993) J. Biol. Chem. 268: 23311-23317) and native (Light andFonseca (1984) J. Biol. Chem. 259: 13195-13198; Matsushima et al. (1994)J. Biol. Chem. 269: 19976-19982) sources. However proteolytic activityfor the other reported membrane-type serine proteases hepsin (Leytus etal. (1988) Biochemistry 27: 1067-1074) and TMPRSS2 (Poloni-Giacobino etal. (1997) Genomics 44: 309-320) are only predicted based upon sequencehomology. In order to produce active recombinant MT-SP 1, a His-taggedfusion of the protease domain was cloned into an E. coli vector andexpressed and purified to homogeneity. Fortuitously, the protease domainrefolded and autoactivated after resuspension and purification frominclusion bodies. This activity, coupled with the lack of activity inthe Ser¹⁹⁵ Ala (Ser⁸⁰⁵ Ala) variant, demonstrates that the cDNA encodesa catalytically proficient protease. Autoactivation of the proteasedomain at the arginine-valine site (Arg⁶¹⁴-Val⁶¹⁵) shows that theprotease has Arg/Lys specificity as predicted by the sequence homologyto other proteases of basic specificity. Specificity and selectivity areconfirmed by the lack of cleavage of AAPX-pNA substrates that do nothave X=R, K. Further characterization with hexahydrotyrosyl-Gly-Arg-pNA(Spectrozyme tPA) revealed an active enzyme with k_(cat)=2.6×10²/s.However, the His-tagged serine protease domain does not cleaveH-Arg-pNA, showing that, unlike trypsin, there is a requirement foradditional subsite occupation for catalytic activity. This suggests thatthe enzyme is involved in a regulatory role that requires selectiveprocessing of particular substrates rather than non-selectivedegradation.

MT-SP1 Function

In other studies, we have found that inhibition of serine proteaseactivity by ecotin or ecotin M84R/M85R inhibits testosterone-inducedbranching ductal morphogenesis and enhances apoptosis in a rat ventralprostate model. Moreover, the rat homolog of MT-SP1 is expressed in thenormal rat ventral prostate (data not shown). Assays of the proteasedomain with ecotin and ecotin M84R/M85R showed that the enzymaticactivity is strongly inhibited (782±92 pM, 9.8±1.5 pM respectively),suggesting that rat MT-SP1 is likely to be inhibited at theconcentrations of these inhibitors used in our experiments. MT-SP1inhibition may result in the observed inhibition of differentiationand/or increased apoptosis. Future studies are aimed at definitivelyresolving the role of MT-SP1 in prostate differentitation. The broadexpression of MT-SP1 in epithelial tissues is consistent with thepossibility that it is involved in cell maintenance or growth anddifferentiation, perhaps by activating growth factors or by processingprohormones. Studies examining the direct role of MT-SP1 indifferentiation and growth of the epithelium in glandular tissues likethe prostate are underway.

MT-SP1 may participate in a proteolytic cascade that results in cellgrowth and or differentiation. Another structurally similarmembrane-type serine protease, enteropeptidase (FIG. 3), is involved ina proteolytic cascade by which activation of trypsinogen leads toactivation of downstream intestinal proteases (5). Enteropeptidase isexpressed only in the enterocytes of the proximal small intestine thusprecisely restricting activation of trypsinogen. Thus, in contrast tosecreted proteases that may diffuse throughout the organism, themembrane association of MT-SP1 should also allow the proteolyticactivity to be precisely localized, which may be important for properphysiological function; improper localization of the enzyme or levels ofdownstream substrates could lead to disease.

We have found subcutaneous coinjection of PC-3 cells with wild-typeecotin or ecotin M84R/M85R led to a decrease in the primary tumor sizecompared to animals in whom PC-3 cells and saline were injected. Sincewild-type ecotin is a poor, micromolar inhibitor of uPA, serineproteases other than uPA likely are involved in this primary tumorproliferation. Both wild-type ecotin and ecotin M84R/M85R are potent,subnanomolar inhibitors of MT-SP1, raising the possibility that MT-SP1plays an important role in progression of epithelial cancers expressingthis protease.

Ecotin injected intraperitoneally also inhibited tumor growth indicatingthat treatment by administration of MT-SP1 modulators can beaccomplished using systemic adminstration.

Direct biochemical isolation of the substrates may be possible if MT-SP1adhesive domains such as the CUB domains or LDLR repeats interact withthe substrates. In addition, likely substrates may be predicted andtested using knowledge of extended enzyme specificity. For example, thecharacterization of the substrate specificity of granzyme B allowed theprediction and confirmation of substrates for this serine protease(Harris et al. (1998) J. Biol. Chem. 273: 27364-27373). Thus, thesecomplimentary studies should further shed light on the physiologicalfunction of this enzyme.

Example 2

Membrane-type serine protease 1 (MT-SP1) was identified as atransmembrane protease expressed by a human prostate cancer cell line,PC-3. We have examined the expression of MT-SP1 in gastric cancertissues and assessed the potential role of this protease in cancerprogression. Western blot and RT-PCR analysis demonstrated exclusiveexpression of MT-SP1 in the cancer tissues of some cases.Immunohistochemically, MT-SP1 was localized in cancer cells, endothelialcells and leukocytes. Because the expression in endothelial cells wasespecially intense, its labeling index (LI) was calculated (0-98,31±5%). MT-SP1 LI was significantly higher in specimens of poorlydifferentiated gastric cancer than in well differentiated cancerspecimens (46±10, 15±6%, respectively, p<0.05). The 11 patients withhigh MT-SP1 expression >=40%) had a lower survival rate than the 21patients with low MT-SP1 expression (<40%) or the 9 patients withoutMT-SP1 expression (p<0.05). These results suggest that MT-SP1 expressionin endothelial cells may play an important role in angiogenesis incancer tissues and is a significant prognostic factor in gastric cancer.

Materials and Methods

Materials.

Surgical specimens of primary tumors obtained from 41 patients whounderwent gastrectomy for gastric cancer between 1985 and 1995 at theSanta Clara Valley Medical Center, San Jose, Calif. and the Palo AltoVeteranis Affair Medical Center, Palo Alto, Calif., were subjected toimmunohistochemical analysis. In addition, surgically resected specimensof gastric cancer and adjacent normal tissue from two patients at theNational Defense Medical College Hospital, Japan were used for Westernand mRNA analysis. Depth of tumor invasion, lymph node involvement,distant metastases and pathologic characteristics were evaluated foreach tumor. TNM classification by the UICC was used for stage grouping(Hermanek and Sobin (1992) UICC TNM classification of malignanttumours., 4th ed. 2nd rev. edition. Berlin: Springer-Verlag). Tumorhistology was divided into three groups: well differentiated, moderatelydifferentiated and poorly differentiated adenocarcinomas.

Western Immunoblotting Analysis.

We compared MT-SP1 expression in cancer tissue with that in adjacentnormal tissue, using immunoblotting analysis. Surgical samples werehomogenized in PBS (phosphate-buffered saline) containing 0.1% TritonX-100. The extracts were electrophoresed on 10% SDS-polyacrylamide gelsthen electrically transferred to polyvinylidene difluoride membrane.Membranes were treated with nonfat milk to reduce nonspecific binding,then incubated for 1 h at room temperature with the primary antibody.After extensive washing, blots were incubated with peroxidase-conjugatedsecond antibodies and developed with detection reagents. Protein contentwas measured using the BCA protein assay and bovine serum albumin as thestandard (Pierce, Rockford, Ill.).

RT-PCR.

mRNA was extracted from each specimen and purified using the RNASTAT-60™ kit (Tel-test, Inc., Friendswood, Tex.). Tissues werehomogenized, lysed, and mRNA extracted and purified according to thevendoris suggested protocol. mRNA was quantified by measuring thespectroscopic absorbance at 260 and 280 nm. RT-PCR was performed withthe Titan™ one tube RT-PCR system according to the manufacturer'sprotocol (Boehringer Mannheim, Indianapolis, Ind.). One mg of templateRNA was reverse transcribed to cDNA in 50 ml of reaction tube with 15 mMMgCl₂, 0.2 mM deoxynucleotide mix, 20 pmol of each MT-SP1 primer, enzymemix at 50° C. for 30 min. The produced cDNA was directly amplified usinga thermal cycler. Initial denaturation was done at 94° C. for 2 minfollowed by 10 and 25 cycles of amplification. The first cycle consistedof 30 s of denaturation at 94° C., 30 s of annealing at 55° C., and 90 sfor enzymatic primer extension at 68° C. The final extension was carriedout at 68° C. for 7 min. The following oligonucleotides were used asRT-PCR primers: MT-SP1-F: 5′-TGC GAC AGT GTG AAC GAC TGC GGA GAC AAC-3′(SEQ ID NO: 32); and MT-SP1-R: 5′-CTC CAC GCT GGA CAG GGG TCC CCC GGAATC-3′ (SEQ ID NO: 33).

As a positive control, we used RNA extract from PC-3 cells (humanprostate cancer cell line). Aliquots (10 mcl) of the RT-PCR produce wereelectrophoresed in 1.5% agarose gel in 1×TAE (40 mM Tris acetate/2 mMsodium EDTA/glacial acetic acid, pH 8.8) containing 0.5 μg-ethidiumbromide.

Immunohistochemistry

Surgical specimens were preserved in a 10% neutralized formaldehydesolution. Each block of paraffin-embedded tumor specimen was cut into 5mm sections and deparaffinized in xylene and ethanol, then immersed in3% hydrogen peroxide-methanol to inhibit endogenous peroxidase. Aftertreatment with normal goat serum, the sections were incubated with a1:100 dilution of rabbit antihuman MT-SP1 antibody overnight at 4° C.They wer washed then treated consecutively with a biotinylated goatanti-rabbit antibody for 1 h. The 3,3′-diaminobenzidine substrate kitand Vectastain Elite ABC kit (Vector Laboratories) were used accordingto the manufacturer's suggested protocol. Counterstaining was performedwith hematoxylin. Negative controls for the immunostaining were carriedout by replacing the primary antibody with preimmune rabbitimmunoglobulin. Vessel rings were counted at 200× magnification. Tenfields were counted for each case, and the proportion of total ringsthat were MT-SP1-positive was defined as the MT-SP1 labeling index(MT-SP LI).

Statistics.

Results shown are the mean±SE. Spearman rank correlation analysis wasused to correlate the degree of each histopathologic factor with MT-SPLI. The cumulative survival rates were calculated by the method ofKaplan-Meier. The survival rates for different groups of patients werecompared by the generalized Wilcoxon test. The specific contribution ofprognostic variables was examined by means of a multivariate Coxisproportional hazard model. A p value of less than 0.05 was consideredstatistically significant.

Results

Immunoblotting for MT-SP1.

Tissue from two cases of stomach cancer were analyzed by Westernimmunoblotting using equal amounts (by weight) of cell lysates fromcancerous and adjacent normal stomach tissue. Using a polyclonalantiserum produced against MT-SP1, the presence of two bands (45 and 100kD), most intense in the tumor tissue of case 2 (FIG. 1), weredemonstrated. These two bands in tumor and adjacent normal tissues fromcase 1 were less intense.

RT-PCR Analysis for MT-SP1.

Analysis of MT-SP1 mRNA from stomach tissues demonstrated the expressionin only tumor tissue of case 2 but not in that of case 1. MT-SP1 was notdetectable by RT-PCR in adjacent normal tissue of either case.

Immunohistochemistry.

We used the immunohistochemical assay to study MT-SP1 expression ingastric cancer tissues. We detected MT-SP1 immunoreactivity in cancercells, the luminal surface of blood vessels, presumably endothelialcells, and some leukocytes. Since the immunoreactivity in blood vesselsin cancer stroma was especially intense, we focused on the correlationbetween MT-SP1 expression in blood vessels and clinicopathologic factorsin gastric cancer.

Relationship Between MT-SP LI and Clinicopathologic Factors.

MT-SP1 expression in blood vessels of cancer stroma did not correlatewith UICC TNM classification. Poorly differentiated tumors showedsignificantly higher MT-SP LI than well differentiated tumors. Tumorswith pT1 showed low MT-SP LI as compared to those with deeper invasionthan submucosa but there were no statistically significant differences.

Relationship Between MT-SP LI and Survival.

There were no significant differences in the overall survival ratesbetween the MT-SP positive group (MT-SP LI>0%) and the MT-SP negativegroup (MT-SP LI=0%). However, when the patients in the MT-SP positivegroup were divided into two groups according to the average index (40%)in MT-SP LI as a cutoff point, eleven patients with a high expression ofMT-SP1 (MT-SP LI>=40%) in blood vessels showed a lower survival ratethan 21 patients with low expression of MT-SP1 (MT-SP LI: 40%) or 9patients with no expression of MT-SP1 (P,0.05). The five-year survivalratge w for patients with an MT-SP LI of 40% or higher was 27.3% versus47.1% for patients with an MT-SP LI lower than 40% or 45.7% for patientswith negative expression of MT-SP1.

Prognostic Analysis of MT-SP1 in Endothelium According to PathologicalStaging.

Kaplan-Meier survival calculations were performed for stages I, II andstages III, IV separately. In stages III, IV, patients (n=30) with anMT-SP LI of 40% or higher had a significantly lower survival rate thanthose (n=11) without MT-SP1 expression in vascular cells of cancertissues (p<0.05). For stages I, II, however, there was no difference insurvival among the three groups.

For multivariate analysis, MT-SP1 expression in blood vessels aroundcancer cells and established risk factors in gastric cancer werecategorized into two classes. MT-SP1 expression was subdivided into twocategories; i) with an MT-SP LI less than 40%, ii) with an MT-SP LI of40% or higher. The depth of tumor invasion and lymph node involvementwere subdivided into two categories; pT1,2 (n=19) and pT3,4 (n=22),pN0,1 (n=27) and pN2 (n=14), respectively. These characteristics wereconfirmed to be significant prognostic determinants in the generalizedWilcoxon test. In Coxis regression model, higher expression of MT-SP1 inblood vessels around cancer cells proved to worsen survivalindependently, following serosal or deeper invasion of primary tumor.

Discussion

MT-SP1 was identified, initially, in a prostate cancer cell line. Webelieve it is involved in homeostatic processes occurring in thepericellular milieu. The cDNA sequence of MT-SP1 encodes a mosaicprotein that contains a transmembrane signal anchor and a serineprotease domain, which is also seen in other membrane-type serineproteases hepsine (Leytus et al. (1988) Biochemistry 27: 1067-1074),enteropeptidase (Kitamoto et al. (1994) Proc Natl Acad Sci USA, 91:7588-7592), and TMPRSS2 (Paoloni-Giacobino et al. (1997) Genomics, 44:309-320). Recently a similar cDNA was cloned from mouse thymic stromalcells which was named epithin (Kim et al. (1999) Immunogenetics 49:420428). An open reading frame was identified that encoded a 902 aminoacid protein with a C-terminal serine protease domain, 4 LDLR domains,and two cub domains. A high level of expression was seen by Northernblotting in mouse intestine and kidney. mRNA was not detected in brain,heart, liver, testis or skeletal muscle. In some tissues, differentforms of mRNA were seen suggesting the possibility of alternativesplicing or alternative polyadenylation sites. Epithin was localized tomouse chromosome 9, ˜16 cM from the centromere.

It is thought that MT-SP1 which is localized to the plasma membranethrough a signal anchor is a component of proteolytic cascades involvedin developmental processes, and physiologic reactions on the surface ofthe gastro-intestinal and genito-urinary epithelium. MT-SP1 is expressedin various malignant epithelial tissues including the digestive andurinary tracts suggesting that it may also have a role in cancerprogression. Recently, Lin et al. reported expression of a similarprotease in human breast cancer cells which was termed, Matriptase (Linet al. (19______) J. Biol. Chem. 274: 18237-18242). A complex of thisprotease with a Kunitz-type inhibitor was also fond in human breast milk(however, since the characterization of its physiological substrate(s)have not been completed, the function of this novel enzyme in cancerprogression is unclear).

In this example, we have clarified a relationship between the expressionof MT-SP1 and clinicopathological factors in gastric cancer. Themolecular weight of the entire MT-SP1 protein is predicted asapproximately 100 kD which is consistent with our immunoblottinganalysis. In this analysis, another band was shown at the molecularweight of 45 kD. This band is likely to represent the activated form ofthe protease domain of MT-SP1 that is released from the prodomain underreducing conditions. Although the expected molecular weight of this formis 28 kD, this difference might be due to glycosylation of the proteasedomain.

Immunohistochemical examination of gastric cancer tissue revealed MT-SP1expression in cancer cells, endothelial cells and some leukocytes. Inthese tissues, endothelial cells showed especially intensive MT-SP1immunoreactivity. This suggests that MT-SP1 plays an important role invascular cells. Although there was not a significant correlation betweenMT-SP1 expression in endothelial cells of gastric cancer tissue andpathological staging, MT-SP1 was highly expressed in the endothelium ofthe poorly differentiated adenocarcinomas. Moreover, overall survivalfor groups of gastric carcinoma patients with highly MT-SP1 expressingendothelium revealed poor prognosis compared to those with low or noMT-SP1. Higher MT-SP1 expression in endothelium was significantlyassociated with lower survival rate. These results suggested that MT-SP1expression in endothelium around cancer cells might be an importantprognostic factor in gastric cancer.

MT-SP1 expression in vessels within the cancer matrix may contribute toangiogenesis in gastric cancer tissue. Some angiogenic factors such asvascular endothelial growth factor (VEGF) (Ferrara et al. (1991) Meth.Enzymol. 198: 391-405, Melnyk (1996) Cancer Res. 56: 921-924) derivedfrom cancer cells might be associated with the MT-SP1 expression inendothelial cells. The interaction between cancer cells and stromalcells in cancer tissue is likely to be important for invasion andmetastasis as described in other reports (Romer et al. (1994) Int JCancer. 57: 553-560; Sieuwerts et al. (1988) Int J Cancer. 76: 829-835).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1-80. (canceled)
 81. An antibody that specifically binds to MT-SP1.
 82. The antibody of claim 81, wherein said antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, and a single-chain antibody.
 83. The antibody of claim 81, wherein said antibody specifically binds to an MT-SP1 serine protease domain.
 84. The antibody of claim 81, wherein said antibody specifically binds to an MT-SP1 serine protease domain having the sequence of amino acids 615 through 855 of SEQ ID NO:2.
 85. A cell line that produces the antibody of claim
 81. 86. A method for evaluating the severity or outcome of a cancer, said method comprising: (a) obtaining a biological sample from a cancer patient having at least a preliminary diagnosis of cancer; and (b) measuring MT-SP1 in said sample by an immunoassay employing the antibody of claim 81 and comparing the sample MT-SP1 level to the MT-SP1 level in a control human wherein a sample MT-SP1 level in excess of MT-S P1 levels in the control human indicates a reduced survival expectancy compared to patients with normal MT-SP1 level.
 87. The method of claim 86, wherein said patient is diagnosed as having a cancer selected from the group consisting of a prostate cancer, a cancer of the digestive tract, a breast cancer, and a urogenital cancer.
 88. The method of claim 86, wherein the level of MT-SP1 is measured by immunohistochemical staining of cells comprising said biological sample.
 89. A method to screen for recurrence of a cancer after removal of a primary tumor, said method comprising: (a) obtaining a biological sample from a cancer patient following removal of a primary tumor; and (b) measuring a level of MT-SP1 in said sample by an immunoassay employing an antibody of claim 81 and comparing the sample MT-SP1 level to the MT-SP1 level in normal healthy humans wherein a sample MT-SP1 level in excess of MT-SP1 levels in normal healthy humans indicates a possible recurrence of said cancer.
 90. The method of claim 89, wherein said method is repeated at a multiplicity of instances after removal of said primary tumor.
 91. A method of monitoring effectiveness of cancer treatment in patients said method comprising: (a) obtaining a first biological sample from said patient prior to or following one or more treatments of a cancer; (b) obtaining a second biological sample from said cancer patient during or after said one or more treatments; and (c) measuring a level of MT-SP1 in said second biological sample by an immunoassay employing an antibody of claim 81 and comparing the level of MT-SP1 in said second sample to the level of MT-SP1 in said first sample, wherein a lower level of MT-SP1 in said second sample as compared to the MT-S P1 level in said first sample indicates efficacy of said one or more treatments
 92. The method of claim 91, wherein said one or more treatments are selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, antihormone therapy, and surgery.
 93. A method of specifically delivering an effector to a tumor cell expressing MT-SP1, said method comprising: (a) providing a chimeric moiety comprising said effector attached to an anti-MT-SP1 antibody of claim 81; and (b) contacting said tumor with said chimeric moiety whereby said chimeric moiety binds to said tumor cell.
 94. The method of claim 93, wherein said tumor cell internalizes at least some of the effector.
 95. The method of claim 93, wherein said effector is selected from the group consisting of a cytotoxin, a detectable label, a radionuclide, a drug, a liposome, a ligand, and an antibody.
 96. The method of claim 93, wherein said effector molecule is cytotoxic.
 97. A chimeric molecule comprising an effector molecule attached to an anti-MT-SP1 antibody of claim
 81. 98. A pharmaceutical composition comprising the chimeric molecule of claim 97 in a pharmaceutically acceptable excipient.
 99. A method of impairing growth of tumor cells expressing an MT-SP1 protein, said method comprising contacting said tumor with a chimeric molecule comprising; an anti-MT-SP1 antibody of claim 81; and an effector molecule wherein said effector molecule is cytotoxic.
 100. A method of treating a cancer in a patient, said method comprising: (a) obtaining a biological sample from a cancer patient having at least a preliminary diagnosis of a cancer, (b) measuring a level of MT-SP1 by immunoassay with an antibody of claim 81 in said sample and comparing the sample MT-SP1 level to the MT-SP1 level in normal healthy humans wherein a sample MT-SP1 level in excess of MT-SP1 levels in normal healthy humans indicates a reduced survival expectancy compared to patients with normal MT-SP1 level; and (c) selecting a patient identified with a MT-SP1 level excess of MT-SP1 levels in normal healthy humans and providing an adjuvant cancer therapy selected from the group consisting of chemotherapy, radiation therapy, reoperation, antihormone therapy, and immunotherapy. 